Vaccine carrier

ABSTRACT

The present invention relates to a hypoallergenic protein consisting of at least one hypoallergenic molecule derived from an allergen, which is fused or conjugated to at least one second non-allergenic protein or fragment thereof.

The present invention relates to novel hypoallergenic molecules and uses thereof.

Type I allergy is an IgE-mediated hypersensitivity disease affecting almost 25% of the population. It is based on the recognition of harmless airborne, insect, venom, food allergen and contact allergen antigens derived from per se harmless antigen sources such as pollen, insects, mold and animal proteins by specific immunoglobulin E. The crosslinking of effector cell-bound IgE antibodies leads to a release of inflammatory mediators (e.g., histamine, leucotrienes) and thus to the immediate symptoms of allergy (e.g., rhinoconjunctivitis, asthma, dermatitis, anaphylaxis). T-cell activation via IgE-dependent as well as IgE-independent mechanisms contributes to chronic allergic inflammation.

The probably only causative forms of allergy treatment is allergen-specific immunotherapy, which is based on the repeated administration of increasing amounts of allergen extracts for most sources. Numerous clinical studies have documented the clinical efficacy of injection immunotherapy and there is evidence for several immunological mechanisms underlying this treatment. Due to the difficulty to prepare high quality allergen extracts for certain allergen sources and the fact that the administration of allergens to patients can cause severe side effects, allergen-specific immunotherapy can only be recommended for certain patients groups and disease manifestations. It is especially difficult to treat patients with co-sensitizations to several different allergen sources and patients suffering from severe disease manifestations such as allergic asthma. Allergic asthma is one of the most vigorous manifestations of allergy, because it severely affects the quality of daily life, causes a high rate of hospitality admissions and can manifest itself in serious, life-threatening forms requiring intensive care of the patient.

Allergen extracts prepared from natural allergen-sources are crude in nature, and it is impossible to influence the quality and amounts of individual allergens in such preparations by technical means. They also contain numerous undefined non-allergenic components, and several recent studies indicate the poor quality of such extracts and document their great heterogeneity.

In the last decade great progress has been made in the field of molecular allergen characterization using recombinant DNA technology. A large number of the most important disease-eliciting allergens has been characterized down to the molecular level, and recombinant allergens mimicking the epitope complexity of natural allergen extracts have been produced. Moreover, several research groups have used the knowledge regarding allergen structures to develop defined new allergy vaccines. Genetic engineering, synthetic peptide chemistry and conjugation of allergens with immunostimulatory DNA sequences have been used to reduce the allergenic activity of the new vaccines and thus the rate of therapy-induced side effects. First promising clinical studies were conducted with such allergen derivatives. Interestingly, it turned out that although IgE-reactivity of genetically engineered recombinant allergens and allergen-derived synthetic T-cell epitope-containing peptides could be strongly reduced or even abolished, these derivatives still could induce systemic side effects appearing several hours after injection. For example, it was reported that T-cell epitope peptides of the major cat allergen, Fel d 1, induced asthma and bronchial hyper reactivity several hours after intracutaneous injection, and there is strong evidence that this effect is T-cell mediated and MHC-restricted.

These results indicate that the removal of IgE-reactivity diminishes IgE-mediated side effects since no immediate reactions were recorded in the course of these immunotherapy studies. However, the allergen-specific T-cell epitopes which have been preserved in the recombinant allergen derivatives as well as in the peptide mixtures are responsible for the late side effects (e.g. very problematic or atopic dermatitis, chronic T cell-mediated allergic skin manifestation). The side effects caused in the case of recombinant allergen-derivatives were relatively mild and in the case of the T-cell peptide vaccines may be overcome by adequate dosing. Both of the two new approaches therefore seem very promising for immunotherapy of allergic rhinoconjunctivitis but may have limitations when it comes to the treatment of severe forms of allergic asthma, where the induction of late side effects in the lung may be very problematic.

In order to administer and consequently to provoke an efficient immune response against peptides, polypeptides and proteins, adjuvants and/or carriers are regularly used. Complete Freund's adjuvant, for instance, is one of the most potent adjuvants available. However, because of its side effects, its use is not approved for humans. Therefore, there exists a need for vaccine compositions able to induce strong immune responses against peptides and polypeptides derived from allergens and of course of other antigens avoiding the use of complete Freund's adjuvant. Further, while BSA has been used successfully as a carrier in animal models it may not be appropriate for use in human vaccine compositions because of the risk of adverse reactions such as the risk of transmitting prion disease (variant Creutzfeldt-Jakob disease). A further challenge to the development of an effective vaccine against allergens is the need for an immune response able to rapidly decrease allergens in an individual or animal. Therefore, high concentrations of allergen-specific antibodies in the blood, which are mainly of the IgG subtype, are needed. In mucosal surfaces IgA antibodies are the primary subtype.

Cholera toxin, a known carrier protein in the art, is also used regularly as an adjuvant, eliminating the need for complete Freund's adjuvant in a vaccine composition. However, cholera toxin increases total and specific IgE antibody levels and leads to IgE-associated inflammatory reactions.

Due to the side effects provoked by most carrier proteins used for vaccination, there exists a need for carrier systems which are able to stimulate immune responses against allergens or other antigens, without using toxic adjuvants, without using poorly tolerated carrier proteins and, in certain situations, without stimulation of potentially pathologic immune responses. Novel carrier systems meeting these specifications can be used towards the formation of novel conjugates and compositions suitable for the treatment or prevention of diseases like allergic diseases.

In Bohle B. et al. (J. Immunol. 172 (11) (2004): 6642-6648) a recombinant fusion protein comprising an S-layer protein moiety and Bet v 1 moiety is described. This molecule comprises the native hyperallergenic Bet v 1 protein.

WO 2004/004761 relates to virus like particles which are fused to an immunogen and which may be used for immunisation.

In WO 2004/003143 the use of fusion proteins comprising a virus like particle and a hyperallergenic molecule as immunogen for vaccination is disclosed.

It is an object of the present invention to provide medicaments and carriers which overcome the aforementioned drawbacks and allow an allergen vaccination with reduced side effects.

Therefore, the present invention relates to a hypoallergenic protein consisting of at least one hypoallergenic molecule derived from an allergen, which is fused or conjugated to at least one second non-allergenic protein or fragment thereof.

In order to provoke an enhanced immune response against a molecule, in particular of a hypoallergenic molecule according to the present invention, said molecule is fused (by genetic engineering) or conjugated (by chemical reactions) to a carrier. A conventional and regularly employed carrier is, for instance, KLH (Keyhole limpet hemocyanin). KLH, which is isolated from the giant sea mollusc Megathura crenulata, is one of the most popular carrier proteins used to create an immunogen for injection. KLH induces a strong antibody response because of its large mass and because it is a non-mammalian protein.

The second protein (the “carrier” or “carrier protein”) to be fused or conjugated to a hypoallergenic molecule of the invention is not derived from an allergen (“non-allergenic”). However, the carrier protein used in the present invention may exhibit T cell reactivity and/or provoke an immune response against itself and the hypoallergenic molecule fused or conjugated to it when administered to an animal or human body. Consequently, if the carrier protein is derived from a pathogen (e.g. virus, bacteria etc.), (protecting) antibodies directed to said carrier and pathogens are produced.

As used herein, “hypoallergenic protein” means a fusion protein/polypeptide of a carrier of a non-allergenic source with a hypoallergenic molecule. Furthermore, a “hypoallergenic protein” is also intended to be a conjugation product (e.g. chemical coupling, adsorption) of a carrier with a hypoallergenic molecule.

“Hypoallergenic” as used herein, refers to molecules with reduced allergenic potential. Such molecules have a decreased capacity to provoke allergic reactions in an individual compared to the wild-type protein from which these molecules are derived.

The at least one hypoallergenic molecule derived from an allergen and fused/conjugated to a second protein is preferably C- and/or N-terminally truncated. “C- and/or N-terminal truncation”, as used herein, means that amino acid residues either from the N-terminus or from the C-terminus or both from the N- and C-terminus of the wild-type allergen are removed by deletion of at least 1, 2, 3, 4, 5, 7, 10, 15, 20, 30 amino acid residues.

The hypoallergenic molecules, i.e. peptides/polypeptides, comprise preferably 10 to 50 amino acids, more preferably 15 to 40 amino acids, in particular 20-30 amino acids and exhibit reduced IgE reactivity. These molecules are designed to exclude T-cell epitopes which may cause T-cell-mediated side effects. T-cell epitopes and molecules exhibiting reduced T-cell response may be determined and identified by methods known by the person skilled in the art (e.g., Bercovici N. et al. Clin Diagn Lab Immunol. (2000) 7:859-864).

It was found that it is possible to design peptide vaccines derived from allergens like the major grass pollen allergens, e.g., Phl p 1, and for the major birch pollen allergen, Bet v 1, using surface exposed peptides. The data obtained show that such peptide vaccines can be produced for any allergen whose primary structure is known according to IgE epitope mapping, three-dimensional structure data or computer-aided prediction of surface-exposed domains. However, the selection of suitable peptides which may be used for vaccination remains crucial, because not all peptides identified with these methods can be employed in vaccination. The peptides suitably used for vaccination purposes should exhibit reduced IgE-binding capacity and—in order to reduce or avoid late side effects—exhibit reduced T-cell reactivity.

The term “derived from an allergen”, as used herein, means that the hypoallergenic molecules according to the present invention are obtained directly from an allergen by fragmentation or truncation. The amino acid sequence of the hypoallergenic molecules of the present invention are preferably at least 80% identical, more preferably at least 90% identical, most preferably at least 95% identical, in particular 100% identical, to the amino sequence stretch of the wild-type allergen, from which the hypoallergenic molecule is derived. However, the molecules which are not 100% identical to the wild-type allergen fragments should be able to bind with at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, strength to an antibody or to antibodies, preferably to IgG antibodies, which are directed to said wild-type allergen fragments.

The degree of identity of a first amino acid sequence to a second amino acid can be determined by a direct comparison between both amino acid sequences using certain algorithms. Such algorithms are, for instance, incorporated in various computer programs (e.g. “BLAST 2 SEQUENCES (blastp)” (Tatusova et al. (1999) FEMS Microbiol. Lett. 174:247-25; Corpet F, Nucl. Acids Res. (1988) 16:10881-10890).

The truncated molecules according to the present invention can be defined as being parts of the complete allergen that induce less activation of allergen-specific T cells than the complete wild-type allergen (preferably at least a 30%, more preferably at least a 50%, most preferably at least a 70%, reduction), exhibit a more than 50% reduced (preferably more than 70%) allergenic activity as evaluated by IgE binding assays and ability to induce IgE-mediated cell activation and when coupled to a carrier as described induce IgG antibodies which inhibit the binding of polyclonal IgE from allergic patients to the complete wild-type allergen.

The peptides should contain sequences from the allergens to avoid overlaps with the mimotopes. Mimotopes, however, which are small peptide mimics (less than 15 amino acids) of antigen pieces and are obtained from random peptide libraries do not represent original, allergen-derived molecules as defined herein. They can not be used according to the invention because they are too small to induce a robust blocking IgG response.

The hypoallergenic molecules according to the present invention may be obtained by recombinant methods or chemical synthesis. Alternatively, it is, of course, also possible to obtain the molecules by enzymatic or chemical cleavage of the wild-type allergen or a polypeptide/protein harbouring the molecule of interest.

The hypoallergenic molecule may comprise preferably at least two truncated allergen molecules derived from at least one allergen, wherein the order of the truncated allergen fragments differs from the order of the fragments in the wild-type allergen if the at least two molecules are derived from the same allergen.

The hypoallergenic molecule according to the present invention may comprise one or more (preferably at least 2, more preferably at least 3) hypoallergenic molecules as defined herein, thus, resulting in a fusion protein. The single hypoallergenic molecules of the fusion protein, which, of course, also lacks IgE-binding capacity and lacks T-cell epitopes, may be derived from allergens of the same and/or of different origin. If the molecules are derived from the same allergen, the order in the hypoallergenic fusion protein should not be identical to the order in the wild-type allergen (this prevents the reconstitution and formation of IgE-binding sites) (see, e.g., WO2004/065414, Linhart B and Valenta R (Int Arch Allergy Immunol. (2004) 134:324-31)).

According to a preferred embodiment of the present invention the at least one hypoallergenic molecule is fused to the N-terminus and/or C-terminus of said at least one second protein or fragment thereof.

The allergen or fragments thereof may be conjugated chemically, e.g., or by recombinant methods to each other. If the allergen or fragment thereof is conjugated chemically to a carrier, said allergen or fragment should be provided with a terminal cysteine residue (resulting in a free sulfhydryl group). To said terminal. (N- or C-terminal) cysteine residue any maleimide-activated carrier protein may be conjugated, thus creating an immunogen/carrier complex. If the allergen or fragment thereof does not have a sulfhydryl group at a terminus, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) chemistry in order to couple amines (lysine) or carboxylic acids (glutamic, aspartic acid or 5′-phosphate) to the carrier protein may be employed.

If the hypoallergenic molecule fused to the N- or C-terminus of the carrier, recombinant methods are employed.

According to a preferred embodiment of the present invention the at least one second protein is a viral, in particular RNA or DNA viral, bacterial fungal or protozoal protein.

The at least one second protein (“carrier”) may be of any of the above-mentioned origin. It is, however, in particular preferred to use proteins which provoke an immune response against the protein itself and the hypoallergenic molecule fused or conjugated thereto. Due to the induction of formation of (protective) antibodies directed also to the at least one second protein, the hypoallergenic protein according to the present invention may also be employed as vaccine for said second protein and its originating source (e.g. virus, bacteria, fungus). Of course it is also possible to use carrier proteins well known in the art (e.g. KLH) as the at least second protein.

The viral protein according to the present invention is preferably a capsid protein.

Viral capsid proteins are especially suited because they induce antiviral activity, provoke the formation of antibodies which block adhesion of viruses, e.g. rhinoviruses, to epithelial cells, exhibit an immuno-modulatory activity towards a Th1 response, increase the immunogenicity of the peptide (i.e., higher anti-peptide and hence higher levels of protective IgG antibodies), are suited and proved for prophylactic vaccination (virus vaccination) and are safe, when capsid proteins are used to whose humans are continuously exposed (e.g. rhinoviruses).

According to another preferred embodiment of the present invention the at least one viral capsid protein is derived from a human pathogenic virus, preferably a virus of the family of picornaviridae.

The virus of the family of picornaviridae is preferably of the genus of rhinoviruses, preferably of the species of human rhinoviruses, in particular human rhinovirus 89 and 14. The capsid protein may be VP1, VP2, VP3 and/or VP4.

The allergen to be fused to a viral capsid protein is preferably selected from the group consisting of major birch pollen allergens, in particular Bet v 1 and Bet v 4, major timothy grass pollen allergens, in particular Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 7, major house dust mite allergens, in particular Der p 1 and Der p 2, major cat allergen Fel d 1, major bee allergens, major wasp allergens, profilins, especially Phl p 12, and storage mite allergens, especially Lep d 2.

Other suited allergens to be used according to the present invention can be derived from the following table.

ALLERGENS Biochem. ID or cDNA (C) or Reference, Species Name Allergen Name Obsolete name MW protein (P) Acc. No. Ambrosia artemisiifolia Amb a 1 antigen E  8 C  8, 20 short ragweed Amb a 2 antigen K  38 C  8, 21 Amb a 3 Ra3  11 C  22 Amb a 5 Ra5  5 C  11, 23 Amb a 6 Ra6  10 C  24, 25 Amb a 7 Ra7  12 P  26 Ambrosia trifida Amb t 5 Ra5G  4.4 C  9, 10, 27 giant ragweed Artemisia vulgaris Art v 1  27-29 C  28 mugwort Art v 2  35 P  28A Art v 3 lipid transfer protein  12 P  53 Art v 4 profilin  14 C  29 Helianthus annuus Hel a 1  34  29A sunflower Hel a 2 profilin  15.7 C Y15210 Mercurialis annua Mer a 1 profilin  14-15 C Y13271 Caryophyllales Chenopodium album Che a 1  17 C AY049012, 29B lamb's-quarters, pigweed, Che a 2 profilin  14 C AY082337 white goosefoot Che a 3 polcalcin  10 C AY082338 Salsola kali Sal k 1  43 P  29C Russian-thistle Rosales Humulus japonicus Hum j 4w C AY335187 Japanese hop Parietaria judaica Par j 1 lipid transfer protein 1  15 C see list of isoallergens Par j 2 lipid transfer protein 2 C see list of isoallergens Par j 3 profilin C see list of isoallergens Parietaria officinalis Par o 1 lipid transfer protein  15  29D B. Grasses Poales Cynodon dactylon Cyn d 1  32 C  30, S83343 Bermuda grass Cyn d 7 C  31, X91256 Cyn d 12 profilin  14 C  31a, Y08390 Cyn d 15  9 C AF517686 Cyn d 22w enolase data pending Cyn d 23 Cyn d 14  9 C AF517685 Cyn d 24 Pathogenesis-related p.  21 P pending Dactylis glomerata Dac g 1 AgDg1  32 P  32 orchard grass Dac g 2  11 C  33, S45354 Dac g 3 C  33A, U25343 Dac g 5  31 P  34 Festuca pratensis Fes p 4w  60 — meadow fescue Holcus lanatus Hol l 1 C Z27084 velvet grass Lolium perenne Lol p 1 group I  27 C  35, 36 rye grass Lol p 2 group II  11 P  37, 37A, X73363 Lol p 3 group III  11 P  38 Lol p 5 Lol p IX, Lol p Ib  31/35 C  34, 39 Lol p 11 hom: trypsin inhibitor  16  39A Phalaris aquatica Pha a 1 C  40, S80654 canary grass Phleum pratense Phl p 1  27 C X78813 timothy Phl p 2 C X75925, 41 Phl p 4 P  41A Phl p 5 Ag25  32 C  42 Phl p 6 C Z27082, 43 Phl p 11 trypsin inhibitor hom.  20 C AF521563, 43A Phl p 12 profilin C X77583, 44 Phl p 13 polygalacturonase  55-60 C AJ238848 Poa pratensis Poa p 1 group I  33 P  46 Kentucky blue grass Poa p 5  31/34 C  34, 47 Sorghum halepense Sor h 1 C  48 Johnson grass C. Trees Arecales Phoenix dactylifera Pho d 2 profilin  14.3 C Asturias p.c. date palm Fagales Alnus glutinosa Aln g 1  17 C S50892 alder Betula verrucosa Bet v 1  17 C see list of birch isoallergens Bet v 2 profilin  15 C M65179 Bet v 3 C X79267 Bet v 4  8 C X87153, S54819 Bet v 6 h: isoflavone reductase  33.5 C see list of isoallergens Bet v 7 cyclophilin  18 P P81531 Carpinus betulus Car b 1  17 C see list of hornbeam isoallergens Castanea sativa Cas s 1  22 P  52 chestnut Cas s 5 chitinase Cas s 8 lipid transfer protein  9.7 P  53 Corylus avellana Cor a 1  17 C see list of hazel isoallergens Cor a 2 profilin  14 C Cor a 8 lipid transfer protein  9 C Cor a 9 11S globulin-like protein  40/? C Beyer p.c. Cor a 10 luminal binding prot.  70 C AJ295617 Cor a 11 7S vicilin-like prot.  48 C AF441864 Quercus alba Que a 1  17 P  54 White oak Lamiales Oleaceae Fraxinus excelsior Fra e 1  20 P  58A, AF526295 ash Ligustrum vulgare Lig v 1  20 P  58A privet Olea europea Ole e 1  16 C  59, 60 olive Ole e 2 profilin  15-18 C  60A Ole e 3  9.2  60B Ole e 4  32 P P80741 Ole e 5 superoxide dismutase  16 P P80740 Ole e 6  10 C  60C, U86342 Ole e 7 ? P  60D, P81430 Ole e 8 Ca2 +− binding protein  21 C  60E, AF078679 Ole e 9 beta-1,3-glucanase  46 C AF249675 Ole e 10 glucosyl hydrolase hom.  11 C  60F, AY082335 Syringa vulgaris Syr v 1  20 P  58A lilac Planteginaceae Plantago lanceolata Pla l 1  18 P P842242 English plantain Pinales Cryptomeria japonica Cry j 1  41-45 C  55, 56 sugi Cry j 2 C  57, D29772 Cupressus arisonica Cup a 1  43 C A1243570 cypress Cupressus sempervirens Cup s 1  43 C see list of common cypress isoallergens Cup s 3w  34 C ref pending Juniperus ashei Jun a 1  43 P P81294 mountain cedar Jun a 2 C  57A, AJ404653 Jun a 3  30 P  57B, P81295 Juniperus oxycedrus Jun o 4 hom: calmodulin  29 C  57C, AF031471 prickly juniper Juniperus sabinoides Jun s 1  50 P  58 mountain cedar Juniperus virginiana Jun v 1  43 P P81825, 58B eastern red cedar Platanaceae Platanus acerifolia Pla a 1  18 P P82817 London plane tree Pla a 2  43 P P82967 Pla a 3 lipid transfer protein  10 P Iris p.c. D. Mites arthropod Acarus siro Aca s 13 fatty acid binding prot.  14* C AJ006774 mite Blomia tropicalis Blo t 1 cysteine protease  39 C AF277840 mite Blo t 3 trypsin  24* C Cheong p.c. Blo t 4 alpha amylase  56 C Cheong p.c. Blo t 5 C U59102 Blo t 6 chymotrypsin  25 C Cheong p.c. Blo t 10 tropomyosin  33 C  61 Blo t 11 paramyosin 110 C AF525465, 61A Blo t 12 St11a C U27479 Blo t 13 Bt6, fatty acid bind prot. C U58106 Blo t 19 anti-microbial pep. hom.  7.2 C Cheong p.c. Dermatophagoides farinae Der f 1 cysteine protease  25 C  69 American house dust mite Der f 2  14 C  70, 70A, see list of isoallergens Der f 3 trypsin  30 C  63 Der f 7  24-31 C SW: Q26456, 71 Der f 10 tropomyosin C  72 Der f 11 paramyosin  98 C  72A Der f 14 mag3, apolipophorin C D17686 Der f 15 98k chitinase  98 C AF178772 Der f 16 gelsolin/villin  53 C  71A Der f 17 Ca binding EF protein  53 C  71A Der f 18w 60k chitinase  60 C Weber p.c. Dermatophagoides microceras Der m 1 cysteine protease  25 P  68 house dust mite Dermatophagoides pteronyssinus Der p 1 antigen P1, cysteine protease  25 C  62, see list of European house dust mite isoallergens Der p 2  14 C  62A-C, see list of isoallergens Der p 3 trypsin  28/30 C  63 Der p 4 amylase  60 P  64 Der p 5  14 C  65 Der p 6 chymotrypsin  25 P  66 Der p 7  22/28 C  67 Der p 8 glutathione transferase C  67A Der p 9 collagenolytic serine pro. P  67B Der p 10 tropomyosin  36 C Y14906 Der p 14 apolipophorin like prot. C Epton p.c. Euroglyphus maynei Eur m 2 C see list of mite isoallergens Eur m 14 apolipophorin 177 C AF149827 Glycyphagus domesticus Gly d 2 C  72B, see isoallergen storage mite list Lepidoglyphus destructor Lep d 2 Lep d 1  15 C  73, 74, 74A, see storage mite isoallergen list Lep d 5 C  75, AJ250278 Lep d 7 C  75, AJ271058 Lep d 10 tropomyosin C  75A, AJ250096 Lep d 13 C  75, AJ250279 Tyrophagus putrescentiae Tyr p 2 C  75B, Y12690 storage mite E. Animals Bos domesticus Bos d 2 Ag3, lipocalin  20 C  76, see isoallergen domestic cattle list (see also foods) Bos d 3 Ca-binding S100 hom.  11 C L39834 Bos d 4 alpha-lactalbumin  14.2 C M18780 Bos d 5 beta-lactoglobulin  18.3 C X14712 Bos d 6 serum albumin  67 C M73993 Bos d 7 immunoglobulin 160  77 Bos d 8 caseins  20-30  77 Canis familiaris Can f 1  25 C  78, 79 (Canis domesticus) Can f 2  27 C  78, 79 dog Can f 3 albumin C S72946 Can f 4  18 P A59491 Equus caballus Equ c 1 lipocalin  25 C U70823 domestic horse Equ c 2 lipocalin  18.5 P  79A, 79B Equ c 3 Ag3-albumin  67 C  79C, X74045 Equ c 4  17 P  79D Equ C 5 AgX  17 P Goubran Botros p.c. Felis domesticus Fel d 1 cat-1  38 C  15 cat (saliva) Fel d 2 albumin C  79E, X84842 Fel d 3 cystatin  11 C  79F, AF238996 Fel d 4 lipocalin  22 C AY497902 Fel d 5w immunoglobulin A 400 Adedoyin p.c. Fel d 6w immunoglobulin M 800- Adedoyin p.c. 1000 Fel. d 7w immunoglobulin G 150 Adedoyin p.c. Cavia poroellus Cav p 1 lipocalin homologue  20 P SW: P83507, 80 guinea pig Cav p 2  17 P SW: P83508 Mus musculus Mus m 1 MDP  19 C  81, 81A mouse (urine) Rattus norvegius Rat n 1  17 C  82, 83 rat (urine) F. Fungi (moulds) 1. Ascomycota 1.1 Dothideales Alternaria alternata Alt a 1  28 C U82633 Alt a 2  25 C  83A, U62442 Alt a 3 heat shock prot.  70 C U87807, U87808 Alt a 4 prot. disulfideisomerase  57 C X84217 Alt a 6 acid ribosomal prot. P2  11 C X78222, U87806 Alt a 7 YCP4 protein  22 C X78225 Alt a 10 aldehyde dehydrogenase  53 C X78227, P42041 Alt a 11 enolase  45 C U82437 Alt a 12 acid ribosomal prot. P1  11 C X84216 Cladosporium herbarum Cla h 1  13  83B, 83C Cla h 2  23  83B, 83C Cla h 3 aldehyde dehydrogenase  53 C X78228 Cla h 4 acid ribosomal prot. P2  11 C X78223 Cla h 5 YCP4 protein  22 C X78224 Cla h 6 enolase  46 C X78226 Cla h 12 acid ribosomal prot. P1  11 C X85180 1.2 Eurotiales Aspergillus flavus Asp fl 13 alkaline serine protease  34  84 Aspergillus fumigatus Asp f 1  18 C M83781, S39330 Asp f 2  37 C U56938 Asp f 3 peroxisomal protein  19 C U20722 Asp f 4  30 C AJ001732 Asp f 5 metalloprotease  40 C Z30424 Asp f 6 Mn superoxide dismut.  26.5 C U53561 Asp f 7  12 C AJ223315 Asp f 8 ribosomal prot. P2  11 C AJ224333 Asp f 9  34 C AJ223327 Asp f 10 aspartic protease  34 C X85092 Asp f 11 peptidyl-prolyl isomeras  24  84A Asp f 12 heat shock prot. P90  90 C  85 Asp f 13 alkaline serine protease  34  84B Asp f 15  16 C AJ002026 Asp f 16  43 C g3643813 Asp f 17 C AJ224865 Asp f 18 vacuolar serine protease  34  84C Asp f 22w enolase  46 C AF284645 Asp f 23 L3 ribosomal protein  44 C  85A, AF464911 Aspergillus niger Asp n 14 beta-xylosidase  105 C AF108944 Asp n 18 vacuolar serine protease  34 C  84B Asp n 25 3-phytase B  66-100 C  85B, P34754 Asp n ?  85 C Z84377 Aspergillus oryzae Asp o 13 alkaline serine protease  34 C X17561 Asp o 21 TAKA-amylase A  53 C D00434, M33218 Penicillium brevicompactum Pen b 13 alkaline serine protease  33  86A Penicillium chrysogenum Pen ch 13 alkaline serine protease  34  87 (formerly P. notatum) Pen ch 18 vacuolar serine protease  32  87 Pen ch 20 N-acetyl glucosaminidas  68  87A Penicillium citrinum Pen c 3 peroxisomal mem. prot.  18  86B Pen c 13 alkaline serine protease  33  86A Pen c 19 heat shock prot. P70  70 C U64207 Pen c 22w enolase  46 C AF254643 Pen c 24 elongation factor 1 beta C AY363911 Penicillium oxalicum Pen o 18 vacuolar serine protease  34  87B 1.3 Hypocreales Fusarium culmorum Fus c 1 ribosomal prot. P2  11* C AY077706 Fus c 2 thioredoxin-like prot.  13* C AY077707 1.4 Onygonales Trichophyton rubrum Tri r 2 C  88 Tri r 4 serine protease C  88 Trichophyton tonsurans Tri t 1  30 P  88A Tri t 4 serine protease  83 C  88 1.5 Saccharomycetales Candida albicans Cand a 1  40 C  89 Cand a 3 peroxisomal protein  29 C AY136739 Candida boidinii Cand b 2  20 C J04984, J04985 2. Basidiomycotina 2.1Hymenomycetes Psilocybe cubensis Psi c 1 Psi c 2 cyclophilin  16  89A Coprinus comatus Cop c 1 leucane zipper protein  11 C AJ132235 shaggy cap Cop c 2 AJ242791 Cop c 3 AJ242792 Cop c 5 AJ242793 Cop c 7 AJ242794 2.2Urediniomycetes Rhodotorula mucilaginosa Rho m 1 enolase  47 C  89B Rho m 2 vacuolar serine protease  31 C AYS47285 2.3 Ustilaginomycetes Malassezia furfur Mala f 2 MF1, peroxisomal  21 C AB011804, 90 membrane protein Mala f 3 MF2, peroxisomal  20 C AB011805, 90 membrane protein Mala f 4 mitochondrial malate dehydrogenase  35 C AF084828, 90A Malassezia sympodialis Mala s 1 C X96486, 91 Mala s 5  18* C AJ011955 Mala s 6  17* C AJ011956 Mala s 7 C AJ011957, 91A Mala s 8  19* C AJ011958, 91A Mala s 9  37* C AJ011959, 91A Mala s 10 heat shock prot. 70  86 C AJ428052 Mala s 11 Mn superoxide dismut.  23 C AJ548421 3. Deuteromycotina 3.1 Tuberculariales Epicoccum purpurascens Epi p 1 serine protease  30 P SW: P83340, 91B (formerly E. nigrum) G. Insects Aedes aegyptii Aed a 1 apyrase  68 C L12389 mosquito Aed a 2  37 C M33157 Apis mellifera Api m 1 phospholipase A2  16 C  92 honey bee Api m 2 hyaluronidase  44 C  93 Api m 4 melittin  3 C  94 Api m 6  7-8 P Kettner p.c. Api m 7 CUB serine protease  39 C AY127579 Bombus pennsylvanicus Bom p 1 phospholipase  16 P  95 bumble bee Bom p 4 protease P  95 Blattella germanica Bla g 1 Bd90k C German cockroach Bla g 2 aspartic protease  36 C  96 Bla g 4 calycin  21 C  97 Bla g 5 glutathione transferase  22 C  98 Bla g 6 troponin C  27 C  98 Periplaneta americana Per a 1 Cr-PII C American cockroach Per a 3 Cr-PI  72-78 C  98A Per a 7 tropomyosin  37 C Y14854 Chironomus kiiensis Chi k 10 tropomyosin  32.5* C AJ012184 midge Chironomus thummi thummi Chi t 1-9 hemoglobin  16 C  99 midge Chi t 1.01 component III  16 C P02229 Chi t 1.02 component IV  16 C P02230 Chi t 2.0101 component I  16 C P02221 Chi t 2.0102 component IA  16 C P02221 Chi t 3 component II-beta  16 C P02222 Chi t 4 component IIIA  16 C P02231 Chi t 5 component VI  16 C P02224 Chi t 6.01 component VIIA  16 C P02226 Chi t 6.02 component IX  16 C P02223 Chi t 7 component VIIB  16 C P02225 Chi t 8 component VIII  16 C P02227 Chi t 9 component X  16 C P02228 Ctenocephalides felis felis Cte f 1 cat flea Cte f 2 Mlb  27 C AF231352 Cte f 3  25 C Thaumetopoea pityocampa Tha p 1  15 P PIR: A59396, 99A pine processionary moth Lepisma saccharina Lep s 1 tropomyosin  36 C AJ309202 silverfish Dolichovespula maculata Dol m 1 phospholipase A1  35 C 100 white face hornet Dol m 2 hyaluronidase  44 C 101 Dol m 5 antigen 5  23 C 102, 103 Dolichovespula arenaria Dol a 5 antigen 5  23 C 104 yellow hornet Polistes annularies Pol a 1 phospholipase A1  35 P 105 wasp Pol a 2 hyaluronidase  44 P 105 Pol a 5 antigen 5  23 C 104 Polistes dominulus Pol d 1 Hoffman p.c. Mediterranean paper wasp Pol d 4 serine protease  32-34 C Hoffman p.c. Pol d 5 P81656 Polistes exclamans Pol e 1 phospholipase A1  34 P 107 wasp Pol e 5 antigen 5  23 C 104 Polistes fuscatus Pol f 5 antigen 5  23 C 106 wasp Polistes gallicus Pol g 5 antigen 5  24 C P83377 wasp Polistes metricus Pol m 5 antigen 5  23 C 106 wasp Vespa crabo Vesp c 1 phospholipase  34 P 107 European hornet Vesp c 5 antigen 5  23 C 106 Vespa mandarina Vesp m 1 Hoffnan p.c. giant asian hornet Vesp m 5 P81657 Vespula flavopilosa Ves f 5 antigen 5  23 C 106 yellowjacket Vespula germanica Ves g 5 antigen 5  23 C 106 yellowjacket Vespula maculifrons Ves m 1 phospholipase A1  33.5 C 108 yellowjacket Ves m 2 hyaluronidase  44 P 109 Ves m 5 antigen 5  23 C 104 Vespula pennsylvanica Ves p 5 antigen 5  23 C 106 yellowjacket Vespula squamosa Ves s 5 antigen 5  23 C 106 yellowjacket Vespula vidua Ves vi 5 antigen 5  23 C 106 wasp Vespula vulgaris Ves v 1 phospholipase A1  35 C 105A yellowjacket Ves v 2 hyaluronidase  44 P 105A Ves v 5 antigen 5  23 C 104 Myrmecia pilosula Myr p 1 C X70256 Australian jumper ant Myr p 2 C S81785 Solenopsis geminata Sol g 2 Hoffman p.c. tropical fire ant Sol g 4 Hoffman p.c. Solenopsis invicta Sol i 2  13 C 110, 111 fire ant Sol i 3  24 C 110 Sol i 4  13 C 110 Solenopsis saevissima Sol s 2 Hoffman p.c. Brazilian fire ant Triatoma protracta Tria p 1 Procalin  20 C AF179004, 111A. California kissing bug H. Foods Gadus callarias Gad c 1 allergen M  12 C 112, 113 cod Salmo salar Sal s 1 parvalbumin  12 C X97824 Atlantic salmon Bos domesticus Bos d 4 alpha-lactalbumin  14.2 C M18780 domestic cattle Bos d 5 beta-lactoglobulin  18.3 C X14712 (milk) Bos d 6 serum albumin  67 C M73993 see also animals Bos d 7 immunoglobulin 160  77 Bos d 8 caseins  20-30  77 Cyprinus carpio Cyp c 1 parvalbumin  12 C 129 (Common carp) Gallus domesticus Gal d 1 ovomucoid  28 C 114, 115 chicken Gal d 2 ovalbumin  44 C 114, 115 Gal d 3 Ag22, conalbumin  78 C 114, 115 Gal d 4 lysozyme  14 C 114, 115 Gal d 5 serum albumin  69 C X60688 Metapenaeus ensis Met e 1 tropomyosin C U08008 shrimp Penaeus aztecus Pen a 1 tropomyosin  36 P 116 shrimp Penaeus indicus Pen i 1 tropomyosin  34 C 116A shrimp Penaeus monodon Pen m 1 tropomyosin  38 C black tiger shrimp Pen m 2 arginine kinase  40 C AF479772, 117 Todarodes pacificus Tod p 1 tropomyosin  38 P 117A squid Helix aspersa Hel as 1 tropomyosin  36 C Y14855, 117B brown garden snail Haliotis midae Hal m 1  49 117C abalone Rana esculenta Ran e 1 parvalbumin alpha  11.9* C AJ315959 edible frog Ran e 2 parvalbumin beta  11.7* C AJ414730 Brassica juncea Bra j 1 2S albumin  14 C 118 oriental mustard Brassica napus Bra n 1 2S albumin  15 P 118A, P80208 rapeseed Brassica rapa Bra r 2 hom: prohevein  25 P81729 turnip Hordeum vulgare Hor v 15 BMAI-1  15 C 119 barley Hor v 16 alpha-amylase Hor v 17 beta-amylase Hor v 21 gamma-3 hordein  34 C 119A, SW: P80198 Secale cereale Sec c 20 secalin see isoall. list rye Triticum aestivum Tri a 18 agglutinin wheat Tri a 19 omega-5 gliadin  65 P PIR: A59156 Zea mays Zea m 14 lipid transfer prot.  9 P P19656 maise, corn Oryza sativa Ory s 1 C 119B, U31771 rice Apium graveolens Api g 1 hom: Bet v 1  16* C Z48967 celery Api g 4 profilin AF129423 Api g 5  55/58 P P81943 Daucus carota Dau c 1 hom: Bet v 1  16 C 117D, see isoallergen carrot list Dau c 4 profilin C AF456482 Corylus avellana Cor a 1.04 hom: Bet v 1  17 C see list of hazelnut isoallergens Cor a 2 profilin  14 C AF327622 Cor a 8 lipid transfer protein  9 C AF329829 Malus domestica Mal d 1 hom: Bet v 1 C see list of apple isoallergens Mal d 2 hom: thaumatin C AJ243427 Mal d 3 lipid transfer protein  9 C Pastorello p.c. Mal d 4 profilin  14.4* C see list of isoallergens Pyrus communis Pyr c 1 hom: Bet v 1  18 C AF05730 pear Pyr c 4 profilin  14 C AF129424 Pyr c 5 hom: isoflavone reductas  33.5 C AF071477 Persea americana Pers a 2 endochitinase  32 C Z78202 avocado Prunus armeniaca Pru ar 1 hom: Bet v 1 C U93165 apricot Pru ar 3 lipid transfer protein  9 P Prunus avium Pru av 1 hom: Bet v 1 C U66076 sweet cherry Pru av 2 hom: thaumatin C U32440 Pru av 3 lipid transfer protein  10 C AF221501 Pru av 4 profilin  15 C AF129425 Prunus domestica Pru d 3 lipid transfer protein  9 P 119C European plum Prunus persica Pru p 3 lipid transfer protein  10 P P81402 peach Pru p 4 profilin  14 C see isoallergen list Asparagus officinalis Aspa o 1 lipid transfer protein  9 P 119D Asparagus Crocus sativus Cro s 1  21 Varasteh A-R p.c. saffron crocus Lactuca sativa Lac s 1 lipid transfer protein  9 Viaths p.c. lettuce Vitis vinifera Vit v 1 lipid transfer protein  9 P P80274 grape Musa × paradisiaca Mus xp 1 profilin  15 C AF377948 banana Ananas comosus Ana c 1 profilin  15 C AF377949 pineapple Ana c 2 bromelain  22.8* C 119E-G, D14059 Citrus limon Cit l 3 lipid transfer protein  9 P Torrejon p.c. lemon Citrus sinensis Cit s 1 germin-like protein  23 P Torrejon p.c. sweet orange Cit s 2 profilin  14 P Torrejon p.c. Cit s 3 lipid transfer protein  9 P Torrejon p.c. Litchi chinensis Lit c 1 profilin  15 C AY049013 litchi Sinapis alba Sin a 1 2S albumin  14 C 120 yellow mustard Glycine max Gly m 1 HPS  7 P 120A soybean Gly m 2  8 P A57106 Gly m 3 profilin  14 C see list of isoallergens Gly m 4 (SAM22) PR-10 prot.  17 C X60043, 120B Vigna radiata Vig r 1 PR-10 protein  15 C AY792956 mung bean Arachis hypogaea Ara h 1 vicilin  63.5 C L34402 peanut Ara h 2 conglutin  17 C L77197 Ara h 3 glycinin  60 C AF093541 Ara h 4 glycinin  37 C AF086821 Ara h 5 profilin  15 C AF059616 Ara h 6 hom: conglutin  15 C AF092846 Ara h 7 hom: conglutin  15 C AF091737 Ara h 8 PR-10 protein  17 C AY328088 Lens culinaris Len c 1 vicilin  47 C see list of lentil isoallergens Len c 2 seed biotinylated prot.  66 P 120C Pisum savitum Pis s 1 vicilin  44 C see list of pea isoallergens Pis s 2 convicilin  63 C pending Actinidia chinensis Act c 2 cysteine protease  30 P P00785 kiwi Act c 2 thaumatin-like protein  24 P SW: P81370, 121 Capsicum annuum Cap a 1w osmotin-like protein  23 C AJ297410 bell pepper Cap a 2 profilin  14 C AJ417552 Lycopersicon esculentum Lyc e 1 profilin  14 C AJ417553 tomato Lyc e 2 b-fructofuranosidase  50 C see isoallergen list Lyc e 3 lipid transfer prot.  6 C U81996 Solanum tuberosum Sola t 1 patatin  43 P P15476 potato Sola t 2 cathepsin D inhibitor  21 P P16348 Sole t 3 cysteine protease inhibitor  21 P P20347 Sole t 4 aspartic protease inhibitor  16 + 4 P P30941 Bertholletia excelsa Ber e 1 2S albumin  9 C P04403, M17146 Brazil nut Ber e 2 11S globulin seed storage protein  29 C AY221641 Juglans nigra Jug n 1 2S albumin  19* C AY102930 black walnut Jug n 2 vicilin-like prot.  56* C AY102931 Juglans regia Jug r 1 2S albumin C U66866 English walnut Jug r 2 vicilin  44 C AF066055 Jug r 3 lipid transfer protein  9 P Pastorello Anacardium occidentale Ana o 1 vicilin-like protein  50 C see isoallergen Cashew list Ana o 2 legumin-like protein  55 C AF453947 Ana o 3 2S albumin  14 C AY081853 Ricinus communis Ric c 1 2S albumin C P01089 Castor bean Sesemum indicum Ses i 1 2S albumin  9 C 121A, AF240005 sesame Ses i 2 2S albumin  7 C AF091841 Ses i 3 7S vicilin-like globulin  45 C AF240006 Ses i 4 oleosin  17 C AAG23840 Ses i 5 oleosin  15 C AAD42942 Cucumis melo Cuc m 1 serine protease  66 C D32206 muskmelon Cuc m 2 profilin  14 C AY271295 Cuc m 3 pathogenesis-rel p. PR-1  16* C P83834 I. Others Anisakis simplex Ani s 1  24 P 121B, A59069 nematode Ani s 2 paramyosin  97 C AF173004 Ani s 3 tropomyosin  41 C 121C, Y19221 Ani s 4  9 P P83885 Argas reflexus Arg r 1  17 C AJ697694 pigeon tick Ascaris suum Asc s 1  10 P 122 worm Carica papaya Car p 3w papain  23.4* C 122A, M15203 papaya Dendronephthya nipponica Den n 1  53 P 122B soft coral Hevea brasiliensis Hev b 1 elongation factor  58 P 123, 124 rubber (latex) Hev b 2 1,3-glucanase  34/36 C 125 Hev b 3  24 P 126, 127 Hev b 4 component of 100- P 128 microhelix complex 115 Hev b 5  16 C U42640 Hev b 6.01 hevein precursor  20 C M36986, p02877 Hev b 6.02 hevein  5 C M36986, p02877 Hev b 6.03 C-terminal fragment  14 C M36986, p02877 Hev b 7.01 hom: patatin from B-serum  42 C U80598 Hev b 7.02 hom: patatin from C-serum  44 C AJ223038 Hev b 8 profilin  14 see list of isoallergens Hev b 9 enolase  51 AJ132580 Hev b 10 Mn superoxide dismut.  26 C see list of isoallergens Hev b 11 class 1 chitinase C see list of isoallergens Hev b 12 lipid transfer protein  9.3 C AY057860 Hev b 13 esterase  42 P P83269 Homo sapiens Hom s 1  73* C Y14314 human autoallergens Hom s 2  10.3* C X80909 Hom s 3  20.1* C X89985 Hom s 4  36* C Y17711 Hom s 5  12.6* C P02538 Triplochiton scleroxylon Trip s 1 class 1 chitinase  38.5 P Kespohl p.c. obeche

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According to a preferred embodiment of the present invention the hypoallergenic molecule exhibits reduced IgE-binding capacity.

According to another preferred embodiment of the present invention the hypoallergenic molecule exhibits reduced T-cell reactivity.

However, also allergen fragments comprising at least one T-cell epitope may be used in the hypoallergenic protein according to the present invention.

“Exhibiting reduced IgE-binding capacity”, as used herein, means that the molecules according to the present invention show significantly reduced IgE-binding capacity or activity (at least 50% less, preferably at least 70% less, more preferably at least 80% less, even more preferably at least 90% less, most preferably at least 95% less, binding capacity compared to the wild-type allergen) or even lack them at all.

IgE-binding activity/capacity of molecules like peptides and proteins can be determined by, for example, an enzyme linked immunosorbent assay (ELISA) using, for example, sera obtained from a subject, (i.e., an allergic subject) that has been previously exposed to the wild-type allergen. Briefly, a peptide to be tested is coated onto wells of a microtiter plate. After washing and blocking the wells, an antibody solution consisting of the plasma of an allergic subject, who has been exposed to the peptide being tested or the protein from which it was derived, is incubated in the wells. A labelled secondary antibody is added to the wells and incubated. The amount of IgE-binding is then quantified and compared to the amount of IgE bound by a purified wild-type allergen.

Alternatively, the binding activity of a peptide can be determined by Western blot analysis. For example, a peptide to be tested is run on a polyacrylamide gel using SDS-PAGE. The peptide is then transferred to nitrocellulose and subsequently incubated with serum from an allergic subject. After incubation with the labelled secondary antibody, the amount of IgE bound is determined and quantified.

Another assay which can be used to determine IgE-binding activity of a peptide is a competition ELISA assay. Briefly, an IgE-antibody pool is generated by combining plasma from allergic subjects who have been shown by direct ELISA to be IgE-reactive with wild-type allergen. This pool is used in ELISA competition assays to compare IgE-binding to wild-type allergen to the peptide tested. IgE-binding for the wild-type allergen and the peptide being tested is determined and quantified.

A “T-cell epitope” means a protein (e.g., allergen) or fragment thereof, for which a T-cell has an antigen specific binding site, the result of binding to said binding site activates the T-cell. The term “exhibiting reduced T-cell reactivity”, as used herein, refers to molecules which exhibit a T-cell reactivity which is significantly reduced compared to the stimulation induced by the wild-type allergen from which the hypoallergenic molecule is derivedusing equimolar amounts in standard assays known in the art (reduced T-cell reactivity means at least 30%, preferably at least 50%, more preferably at least 70%, most preferably at least 90%, less stimulation of hypoallergenic molecules compared to the wildtype allergen at equimolar amounts). In a particular preferred embodiment of this invention, the molecules may “lack” T-cell epitopes and thus molecule shows reduced T-cell reactivity in the individual(s) to be treated (i.e., who is to receive an epitope-presenting valency platform molecule). It is likely that, for example, an allergen-derived molecule may lack a T-cell epitope(s) with respect to an individual, or a group of individuals, while possessing a T-cell epitope(s) with respect to other individual(s). Methods for detecting the presence of a T-cell epitope are known in the art and include assays which detect T-cell proliferation (such as thymidine incorporation). Immunogens that fail to induce statistically significant incorporation of thymidine above background (i.e., generally p less than 0.05 using standard statistically methods) are generally considered to lack T-cell epitopes, although it will be appreciated that the quantitative amount of thymidine incorporation may vary, depending on the immunogen being tested (see, e.g., Zhen L. et al. (Infect Immun. (2003) 71:3920-3926)). Generally, a stimulation index below about 2-3, more preferably less than about 1, indicates lack of T-cell reactivity and epitopes. The presence of T-cell epitopes can also be determined by measuring secretion of T-cell-derived lymphokines according to standard methods. The stimulation index (SI) may be calculated by dividing the proliferation rate (Thymidine uptake) of stimulated cells through the proliferation rate of unstimulated cells in medium alone. SI=1 means no stimulation, SI<1 indicates toxic effects and SI>1 indicates stimulation of cells. Location and content of T-cell epitopes, if present, can be determined empirically.

The cytokine secretion may be determined in addition to the stimulation of T cells. For example, IFN-gamma has been recognized as a harmful cytokine. Other examples may be TNF-alpha, IL-5, IL-4, IL-8 etc.

The allergen fragment is preferably composed of amino acids 151 to 177, 87 to 117, 1 to 30, 43 to 70 or 212 to 241 of Phl p 1, amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58, 132 to 162, 217 to 246, 252 to 283 or 176 to 212 of Phl p 5, amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1, amino acids 30 to 59, 50 to 79 or 75 to 104 of Bet v 1, amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1-35, 36-70, 71-110, 111-145, 140-170, 175-205, 210-250 or 250-284 of Der p 10, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70 of Clone 30, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.

The specific amino acid sequences of the above identified allergen-derived molecules are:

Peptide Position Sequence SEQ ID NO:  Pep Alt a 1.1  19-58 APLESRQDTASCPVTTEDGYVWKISEFYGRKPEG-  23 TYYNSL Pep Alt a 1.2  59-95 GFNIKATNGGTLDFTCSAQADKLEDHKWYSCGENS  24 FM Pep Alt a 1.3  91-120 ENSFMDFSFDSDRSGLLLKQKVSDDITYVA  25 Pep Alt a 1.4 121-157 TATLPNYCRAGGNGPKDFVCQGVADAYITLVTLPK  26 SS Pep Alt a 2.1   1-40 MHSSNNFFKDNIFRSLSKEDPDYSRNIEGQVIRLH-  27 WDWAQ Pep Alt a 2.2  41-80 LLMLSAKRMKVAFKLDIEKDQRVWDRCTADDLK-  28 GRNGFKR Pep Alt a 2.3  81-120 CLQFTLYRPRDLLSLLNEAFFSAFRENRETIINTD-  29 LEYAA Pep Alt a 2.4 121-160 KSISMARLEDLWKEYQKIFPSIQVITSAFRSIE-  30 PELTVYT Pep Alt a 2.5 161-190 CLKKIEASFELIEENGDPKITSEIQLLKAS  31 Pep Alt a 6.1   1-34 MTITKIHARSVYDSRGNPTVEVDIVTETGLHRAI  32 Pep Alt a 6.2  35-74 VTETGLHRAIVPSGASTGSHEACELRDGDKSKWG-  33 GKGVTK Pep Alt a 6.3  74-115 APALIKEKLDVKDQSAVDAFLNKLDGTTNKTNL-  34 GANAILGVS Pep Alt a 6.4 125-165 EKGVPLYAHISDLAGT KKPYVLPVPF QNVLNG-  35 GSHAGGRLA Pep Alt a 6.5 174-213 CEAPTFSEAMRQGAEVYQKLKALAKKTYGQSAGN-  36 VGDEGG Pep Alt a 6.6 241-280 IKIAMDVASSEFYKADEKKYDLDFKNPDSDKSKWL-  37 TYEQL Pep Alt a 6.7 294-333 VSIEDPFAEDDWEAWSYFFKTYDGQIVGDDLTVT-  38 NPEFIK Pep Alt a 6.8 361-400 AKDAFGAGWGMVSHRSGETEDVTIADIVVGLRS-  39 GQIKTG Pep Alt a 6.9 401-438 APARSERLAKLNQILRIEEELGDNAVYAGNNFR-  40 TAVNL Pep Amb a 1.1  31-70 EILPVNETRRLTTSGAYNIIDGCWRGKADWAEN-  41 RKALADC Pep Amb a 1.2  80-120 GGKDGDIYTVTSELDDDVANPKEGTLRFGAAQNR-  42 PLWIIFE Pep Amb a 1.3 125-155 IRLDKEMVVNSDKTIDGRGAKVEIINAGFTL  43 Pep Amb a 1.4 160-200 NVIIHNINMHDVKVNPGGLIKSNDGPAAPRAGSDG-  44 DAISIS Pep Amb a 1.5 225-263 GTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGMLAT-  45 VAF Pep Amb a 1.6 264-300 NTFTDNVDQRMPRCRHGFFQVVNNNYDKWGSYAIGGS  46 Pep Amb a 1.7 305-350 ILSQGNRFCARDERSKKNVLGRHGEAAAESMKWN-  47 WRTNKDVLENGA Pep Amb a 1.8 356-396 GVDPVLTPEQSAGMIPAEPGESALSLTSSAGVLSC-  48 QPGAPC Pep Art v 1.1   27-70 SKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACH-  49 KREAGKES Pep Art v 1.2  70-100 SCFCYFDCSKSPPGATPAPPGAAPPPAAGGS  50 Pep Art v 1.3  92-132 APPPAAGGSPSPPSDGGSPPPPADGGSPPVDGG-  51 SPPPPSTH Can f 1 Pep 1  19-56 QDTPALGKDTVAVSGKWLKAMTADQEVPEKPDSVT-  52 P, Can f 1 Pep 2  51-90 DSVTPMILKAQKGGNLEAKITMLTNGQCQNITVVL-  53 HKTSE Can f 1 Pep 3  78-118 CQNITVVLHKTSEPGKYTAYEGQRVVFIQPSPVRD-  54 HYILYC Can f 1 Pep 4 106-145 QPSPVRDHYILYCEGELHGRQIRMAKLLGRD-  55 PEQSQEALE Can f 1 Pep 5 135-174 RDPEQSQEALEDFREFSRAKGLNQEILELAQSETC-  56 SPGGQ Can f 2 Pep 1  19-58 QEGNHEEPQGGLEELSGRWHSVALASNKADLIKP-  57 WGHFRV Can f 2 Pep 2  52-91 PWGHFRVFIHSMSAKDGNLHGDILIPQDGQCEK-  58 VSLTAFK Can f 2 Pep 3  82-119 CEKVSLTAFKTATSNKFDLEY-  59 WGHNDLYAEVDPKSYL Can f 2 Pep 4 106-144 NDLYLAEVDPKSYLILYMINQYN-  60 DDTSLVAHLMVRDLSR Can f 2 Pep 5 139-180 VRDLSRQQDFLPAFESVCEDIGLHKDQIVVLS-  61 DDDRCQGSRD Fel d 2 Pep 1  25-58 EAHQSEIAHRFNDLGEEHFRGLVLVAFSQYLQQC  62 Fel d 2 Pep 2  99-133 CTVASLRDKYGEMADCCEKKEPERNECFLQHKDDN  63 Fel d 2 Pep 3 154-183 NEQRFLGKYLYEIARRHPYFYAPELLYYAE  64 Fel d 2 Pep 4 277-307 CADDRADLAKYICENQDSISTKLKECCGKPV  65 Fel d 2 Pep 5 334-363 VEDKEVCKNYQEAKDVFLGTFLYEYSRRHP  66 Fel d 2 Pep 6 373-402 LAKEYEATLEKCCATDDPPACYAHVFDEFK  67 Fel d 2 Pep 7 544-573 EKQIKKQSALVELLKHKPKATEEQLKTVMG  68 Fel d 2 Pep 8 579-608 VDKCCAAEDKEACFAEEGPKLVAAAQAALA  69 Fel d 2 Pep 9  58-99 CPFEDHVKLVNEVTEFAKGCVADQSAANCEK-  70 SLHELLGDKLC Fel d 2 Pep 10 125-165 CFLQHKDDNPGFGQLVTPEADAMCTAFHENEQRFLG-  71 KYLYE Fel d 2 Pep 11 183-224 EEYKGVFTECCEAADKAACLTPKVDALREKVLAS-  72 SAKERLKC Fel d 2 Pep 12 224-261 CASLQKFGERAFKAWSVARLSQKFPKAE-  73 FAEISKLVTD Fel d 2 Pep 13 252-289 FAEISKLVTDLAHIHKECCHGDLLECADDRADLAKY-  74 IC Fel d 2 Pep 14 303-340 CGKPVLEKSHCISEVERDELPADLPPLAVD-  75 FVEDKEVC Fel d 2 Pep 15 416-457 CELFEKLGEYGFQNALLVRYTKKVPQVST-  76 PTLVEVSRSLGKV Fel d 2 Pep 16 460-500 CTHPEAERLSCAEDYLSVVLNRLCVLHEKTPVSER-  77 VTKC Fel d 2 Pep 17 501-542 CTESLVNRRPCFSALQVDETYVPKEFSAETFTF-  78 HADLCTLPE Pep Ole e 1.1   1-40 EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIP-  79 GASLR Pep Ole e 1.2  36-66 GASLRLQCKDKENGDVTFTEVGYTRAEGLYS  80 Pep Ole e 1.3  63-99 GLYSMLVERDHKNEFCEITLISSGRKDCNEIPTEGWA  81 Pep Ole e 1.4  86-120 GRKDCNEIPTEGWAKPSLKFKLNTVNGTTRTVNPL  82 Pep Ole e 1.5 107-145 LNTVNGTTRTVNPLGFFKKEALPKCAQVYNKL-  83 GMYPPNM Pep Par j 2.1  31-60 GEEACGKVVQDIMPCLHFVKGEEKEPSKEC  84 Pep Par j 2.2  45-80 CLHFVKGEEKEPSKECCSGTKKLSEEVKTTEQKREA  85 Pep Par j 2.3  60-96 CCSGTKKLSEEVKTTEQKREACKCIVRATKGISGIKN  86 Pep Par j 2.4  97-133 ELVAEVPKKCDIKTTLPPITADFDCSKIQSTIFRGYY  87 Der p 1 Pep 1   1-30 TNACSINGNAPAEIDLRQMRTVTPIRMQGG  88 Der p 1 Pep 2  52-94 NQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQ  89 Der p 1 Pep 3  85-115 HNGVVQESYYRYVAREQSCRRPNAQRFGISN  90 Der p 1 Pep 4  99-135 REQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTH  91 Der p 1 Pep 5 145-175 KDLDAFRHYDGRTIIQRDNGYQPNYHAVNIV  92 Der p 1 Pep 6 155-187 GRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWI  93 Der p 1 Pep 7 175-208 VGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI  94 Der p 1 Pep 8 188-222 VRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVIL  95 Der p 2 Pep 1   1-33 DQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGK  96 Der p 2 Pep 2  21-51 CHGSEPCIIHRGKPFQLEAVFEANQNSKTAK  97 Der p 2 Pep 3  42-73 EANQNSKTAKIEIKASIEGLEVDVPGIDPNAG  98 Der p 2 Pep 4  62-103 EVDVPGIDPNACHYMKCPLVKGQQYDIKYTWIVP-  99 KIAPKSEN Der p 2 Pep 5  98-129 APKSENVVVTVKVMGDNGVLACAIATHAKIRD 100 Der p 5 Pep 1   1-35 MEDKKHDYQNEFDFLLMERIHEQIKKGELALFYLQ 101 Der p 5 Pep 2  25-60 KKGELALFYLQEQINHFEEKPTKEMKDKIVAEMDTI 102 Der p 5 Pep 3  65-95 DGVRGVLDRLMQRKDLDIFEQYNLEMAKKSG 103 Der p 5 Pep 4  78-114 DLDIFEQYNLEMAKKSGDILERDLKKEEARVKKIEV 104 Der p 7 Pep 1   1-30 DPIHYDITEEINKAVDEAVAAIEKSETFD 105 Der p 7 Pep 2  20-50 VAAIEKSETFDPMKVPDHSDKFERHIGIIDL 106 Der p 7 Pep 3  50-80 LKGELDMRNIQVRGLKQMKRVGDANVKSEDG 107 Der p 7 Pep 4  90-125 VHDDVVSMEYDLAYKLGDLHPNTHVISDIQDFVVEL 108 Der p 7 Pep 5 125-155 LSLEVSEEGNMTLTSFEVRQFANVVNHIGGL 109 Der p 7 Pep 6 165-198 LSDVLTAIFQDTVRAEMTKVLAPAFKKELERNNQ 110 Der p 10 Pep 1   1-35 MEAIKKKMQAMKLEKDNAIDRAEIAEQKARKANLR 111 Der p 10 Pep 2  36-70 AEKSEEEVRALQKKIQQIENELDQVQEQLSAANTK 112 Der p 10 Pep 3  71-110 LEEKEKALQTAEGDVAALNRRIQLEEDLERSEER- 113 LKIAT Der p 10 Pep 4 111-145 AKLEEASQSADESERMRKMLEHRSITDEERMEGLE 114 Der p 10 Pep 5 140-170 RMEGLENQLKEARMMAEDADRKYDEVARKLA 115 Der p 10 Pep 6 175-205 DLERAEERAETGESKIVELEEELRVVGNNLK 116 Der p 10 Pep 7 210-250 SEEKAQQREEAHEQQIRIMTTKLKEAEARAEFAERS- 117 VQKLQ Der p 10 Pep 8 250-284 QKEVDRLEDELVHEKEKYKSISDELDQTFAELTGY 118 Der p 21 Per 1   1-35 MFIVGDKKEDEWRMAFDRLMMEELETKIDQVEKGL 119 Der p 21 Per 2  35-72 LHLSEQYKELEKTKSKELKEQILRELTIGENFMKGAL 120 Der p 21 Per 3  70-100 GALKFFEMEAKRTDLNMFERYNYEFALESIK 121 Der p 21 Per 4  90-122 YNYEFALESIKLLIKKLDELAKKVKAVNPDEYY 122 Clone 30 Pep 1   1-32 MANDNDDDPTTTVHPTTTEQPDDKFECPSRFG 123 Clone 30 Pep 2  15-48 PTTTEQPDDKFECPSRFGYFADPKDPHKFYICSN 124 Clone 30 Pep 3  32-70 GYFADPKDPHKFYICSNWEAVHKDCPGNTRWNEDEE 125 TCT Bet v 1 Pep 1  30-59 LFPKVAPQAISSVENIEGNGGPGTIKKISF 126 Bet v 1 Pep 2  50-79 GPGTIKKISFPEGFPFKYVKDRVDEVGHTN 127 Bet v 1 Pep 3  75-104 VDHTNFKYNYSVIEGGPIGTLEKISNEIK 128 Fel d 1 chain   1-34 EICPAVKRDVDLFLTGTPDEYVEQVAQKALPVVC 129 1 Pep 2 Fel d 1 chain  35-70 LENARILKNCVDAKMTEEDKENALSLLDKIYTSPLC 130 1 Pep 2 Fel d 1 chain   1-34 VKMAITCPIFYDVFFAVANGNELLLDLSLTKVNAC 131 2 Pep 1 Fel d 1 chain  35-63 TEPERTAMKKIQDCYVENGLISRVLDGLVC 132 2 Pep 2 Fel d 1 chain  64-92 CMTTISSSKDCMGEAVQNTVEDLKLNTLGR 133 2 Pep 3 Phl p 5 Pep 1  98-128 CGAASNKAFAEGLSGEPKGAAESSSKAALTSK 134 Phl p 5 Pep 2  26-58 ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKC 135 Phl p 5 Pep 3 132-162 AYKLAYKTAEGATPEAKYDAYVATLSEALRIC 136 Phl p 5 Pep 4 217-246 CEAAFNDAIKASTGGAYESYKFIPALEAAVK 137 Phl p 5 Pep 5 252-283 TVATAPEVKYTVFETALKKAITAMSEAQKAAKC 138 Phl p 5 Pep 6 176-212 CAEEVKVIPAGELQVIEKVDAAFK- 139 VAATAANAAPANDK Phl p 5 Pep 1a  93-128 CFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK 141 Phl p 5 Pep 2b  26-53 ADLGYGPATPAAPAAGYTPATPAAPAEAC 142

The terms “fragments thereof” and “sequence variations thereof” refer to peptides which are deduced from the allergen-derived molecules disclosed herein and show biochemical properties (e.g. the capacity to prevent IgE binding to the allergen from which those molecules are derived from) which are comparable or identical to said allergen-derived molecules. The fragments of the present invention comprise at least 5, preferably at least 7, more preferably at least 10, successive and/or a maximum of 95%, preferably a maximum of 90%, more preferably a maximum of 80% amino acid residues of the allergen-derived molecule. The term “sequence variation” includes modifications of the peptides such as fragmentation (see above), amino acid substitutions (e.g. with other natural or non-natural amino acids or amino acid derivatives), deletions or additions. “Sequence variation” refers also to said allergen-derived molecules of the above table, wherein at least 1, preferably at least 2, more preferably at least 3, even more preferably at least 4 (5, 6, 7, 8, 9, 10, 15, 20) amino acid residues are added to the C- and/or N-terminus.

It is noted that the clone 30 allergen is an allergen derived from the house dust mite Dermatophagoides pteronyssinus and consists of the following sequence:

MANDNDDDPTTTVHPTTTEQP- DDKFECPSRFGYFADPKDPHKFYICSNWEAVHKDCPGNTRWNEDEETCT (SEQ ID No. 140; see also AT A 733/2006).

According to the present invention also peptides are encompassed which are at least 80% identical, preferably 90% identical, to the amino sequences disclosed above.

Another aspect of the present invention relates to a nucleic acid molecule encoding a fused hypoallergenic protein according to the present invention.

Another aspect of the present invention relates to a vector comprising a nucleic acid molecule according to the present invention.

Said vector is preferably an expression vector.

The vector harbouring the nucleic acid molecule of the present invention may be used for cloning purposes or for the production of expression vectors. Said vector can be a plasmid, cosmid, virus, bacteriophage or any other vector commonly used in genetic engineering, and can include, in addition to the nucleic acid molecule of the invention, eukaryotic or prokaryotic elements for the control of the expression, such as regulatory sequences for the initiation and the termination of the transcription and/or translation, enhancers, promoters, signal sequences and the like.

According to a preferred embodiment of the present invention the vector is a bacterial, fungal, insect, viral or mammalian vector.

The vector of the present invention may preferably be employed for cloning and expression purposes in various hosts. Therefore, said vector comprises besides a nucleic acid encoding for a hypoallergenic molecule or fusion protein according to the present invention host specific regulatory sequences.

Another aspect of the present invention relates to a host comprising a nucleic acid molecule or a vector according to the present invention.

The nucleic acid molecule and the vector according to the present invention may be introduced into a suitable host. Said molecule may be incorporated into the genome of the host. The vector may exist extrachromosomally in the cytoplasm or incorporated into the chromosome of the host.

Yet another aspect of the present invention relates to an antibody directed against a hypoallergenic molecule, hypoallergenic fusion protein or a fusion protein according to the present invention.

According to a preferred embodiment of the present invention the antibody is a monoclonal or polyclonal antibody.

Antibodies according to the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments and epitope-binding fragments of any of the above. Furthermore, antibodies are considered to be immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention are preferably of the types IgG, IgM, IgD, IgA and IgY, class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Polyclonal antibodies can be prepared by administering a polypeptide of the invention, preferably using an adjuvant, to a non-human mammal and collecting the resultant antiserum. Improved titers can be obtained by repeated injections over a period of time. There is no particular limitation to the species of mammals which may be used for eliciting antibodies; it is generally preferred to use rabbits or guinea pigs, but horses, cats, dogs, goats, pigs, rats, cows, sheep, camels etc., can also be used. In the production of antibodies, a definite amount of immunogen of the invention is, e.g., diluted with physiological saline solution to a suitable concentration, and the resulting diluted solution is mixed with, e.g., complete Freund's adjuvant to prepare a suspension or with mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. The suspensions and mixtures are administered to mammals, e.g., intraperitoneally, e.g., to a rabbit, using from about 50 μg to about 2,500 μg polypeptide of the invention per administration. The suspension is preferably administered about every two weeks over a period of up to about 2-3 months, preferably about 1 month, to effect immunization. The antibody is recovered by collecting blood from the immunized animal after the passage of 1 to 2 weeks after the last administration, centrifuging the blood and isolating serum from the blood.

Monoclonal antibodies may, e.g., be of human or murine origin. Murine monoclonal antibodies may be prepared by the method of Köhler and Milstein (Köhler, G. and Milstein, C., Nature 256 (1975) 495), e.g., by fusion of spleen cells of hyperimmunized mice with an appropriate mouse myeloma cell line.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. No. 5,807,715; U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397.

Humanized antibodies are antibody molecules from a non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modelling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; WO 91/09967; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,530,101; and U.S. Pat. No. 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-913 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

The antibodies according to the present invention may advantageously be used for desensitization of an individual suffering from an allergy, in particular from house dust mite allergy. For passive immunisation the antibody is preferably an IgG or a derivative thereof (e.g., chimeric or humanized antibody). Furthermore, this antibody may also be used for desensibilisation of an individual.

Another aspect of the present invention relates to a vaccine formulation comprising a hypoallergenic protein or an antibody according to the present invention.

The vaccine formulation according to the present invention may be formulated as known in the art and necessarily adapted to the way of administration of said vaccine formulation.

Preferred ways of administration of the vaccine formulation (of the present invention) include all standard administration regimes described and suggested for vaccination in general and allergy immunotherapy specifically (orally, transdermally, intravenously, intranasally, via mucosa, rectally, etc). However, it is particularly preferred to administer the molecules and proteins according to the present invention subcutaneously or intramusculary.

The vaccine formulation according to the present invention may only comprise a viral capsid protein or fragments thereof of a member of the genus of rhinovirus

Said formulation preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.

In order to increase the immunogenicity of the hypoallergenic molecules according to the present invention, adjuvants, for instance, may be used in a medicament according to the present invention. An adjuvant according to the present invention is an auxiliary agent which, when administered together or in parallel with an antigen, increases its immunogenicity and/or influences the quality of the immune response. Hence, the adjuvant can, e.g., considerably influence the extent of the humoral or cellular immune response. Customary adjuvants are, e.g., aluminum compounds, lipid-containing compounds or inactivated mycobacteria.

Generally, adjuvants can be of different forms, provided that they are suitable for administration to human beings. Further examples of such adjuvants are oil emulsions of mineral or vegetal origin, mineral compounds such as aluminium phosphate or hydroxide, or calcium phosphate, bacterial products and derivatives, such as P40 (derived from the cell wall of Corynebacterium granulosum), monophosphoryl lipid A (MPL, derivative of LPS) and muramyl peptide derivatives and conjugates thereof (derivatives from mycobacterium components), alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, etc. (see, e.g., Gupta R. K. et al. (Vaccine 11:293-306 (1993)) and Johnson A. G. (Clin. Microbiol. Rev. 7:277-289).

According to another preferred embodiment of the present invention said formulation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule or antibody.

Another aspect of the present invention relates to the use of a hypoallergenic protein or an antibody according to the present invention for manufacturing a medicament for the treatment or prevention of a viral infection and/or an allergy in a human or animal.

Said medicament preferably further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.

The medicament according to the present invention may be used for active (administration of the hypoallergenic protein and/or molecules of the invention) as well as for passive immunization (antibodies directed to the hypoallergenic protein and/or molecules of the invention).

According to a preferred embodiment of the present invention said medicament comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule, nucleic acid molecule, vector, host or antibody.

The medicament is preferably administered to an individual in amount of 0.01 mg/kg body weight to 5 mg/kg body weight, preferably 0.1 mg/kg body weight to 2 mg/kg body weight.

The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Empirical considerations, such as the half life, will generally contribute to determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy.

The individual to whom the medicament according to the present invention is administered is preferably an individual or animal which is at risk to become an allergy.

Subjects having or at risk of having an allergic condition, disorder or disease include subjects with an existing allergic condition or a known or a suspected predisposition towards developing a symptom associated with or caused by an allergic condition. Thus, the subject can have an active chronic allergic condition, disorder or disease, an acute allergic episode, or a latent allergic condition, disorder or disease. Certain allergic conditions are associated with seasonal or geographical environmental factors. Thus, at risk subjects include those at risk from suffering from a condition based upon a prior personal or family history, and the season or physical location, but which the condition or a symptom associated with the condition may not presently manifest itself in the subject.

The administration of the medicament according to the present invention, which comprises at least one hypoallergenic molecule as described herein, to an individual may prevent sensitization of said individual or may induce an appropriate immune response to allergens. If the medicament of the present invention is used to prevent sensitization, it should be administered to an individual prior to the first contact with said allergen. Therefore, it is preferred to administer the medicament according to the present invention to neonates and children. It turned out that also the administration of the medicament according to the present invention to pregnant individuals will induce the formation of antibodies directed against allergens in the unborn child. It is especially beneficiary to use hypoallergenic molecules according to the present invention for such therapies, because due to the lack of T-cell epitopes side effects occurring in the course of allergen immunotherapy can significantly be reduced or even be completely avoided.

Yet another aspect of the present invention relates to the use of a hypoallergenic protein or an antibody according to the present invention for the diagnosis of an allergy and/or a viral infection in an individual.

Another aspect of the present invention relates to the use a viral capsid protein from a virus of the family of picornaviridae as a carrier in medicaments or vaccines or for diagnosing a viral infection, in particular common cold.

As a valuable alternative to the widely spread KLH carrier protein viral capsid proteins of viruses of the family of picornaviridae may be used. The carrier may be conjugated chemically or fused with recombinant techniques to peptides, proteins and polypeptides or other antigens. Furthermore, the viral capsid protein may be used to detect, e.g., antibodies directed to said capsid protein in the serum of an individual.

One of the advantages of such a carrier is that not only the antigen fused or conjugated thereon may be exposed to the immune system, but also an immune response against the capsid protein of a rhinovirus is induced. Consequently, such a vaccination leads to the prevention and/or treatment of diseases caused by rhinoviruses. The virus is preferably of the species of human rhinoviruses, in particular human rhinovirus 89 and 14.

Another aspect of the present invention relates to a hypoallergenic molecule derived from Phl p 5 (Genbank Nr. X7435) having a C- and/or N-terminal truncation and lacking substantially IgE-binding capacity.

Grass pollen is one of most potent outdoor seasonal sources of airborne allergens responsible for hay fever and allergic asthma.

More than 40% of allergic individuals display IgE-reactivity with grass pollen allergens, which are divided into more than 11 groups. More than 80% of the grass pollen allergic patients react with group 5 allergens.

Group 5 allergens are non-glycosylated, highly homologous proteins with a molecular mass range from 25-33 kD. Several group 5 allergens have been cloned and/or immunologically characterized.

The trial to reduce the allergenic activity by introducing pointmutations, mutations of several amino acids in row or deletions showed no effect (Schramm G, et al. J Immunol 1999; 162: 2406-1435). IgE-binding regions of Phl p 5 (Flicker S, et al. J Immunol 2000; 165: 3849-3859) have already been described and the three-dimensional structure has been solved (Maglio O, et al. 2002. Protein Eng. 15:635-642).

It turned out that in particular the Phl p 5 peptides according to the present invention, which are C- and/or N-terminally truncated and lack IgE-binding capacity, may be employed for the active vaccination of individuals.

According to a preferred embodiment of the present invention the truncated molecule lacks T-cell epitopes.

As already outlined above, late side effects of allergen immunotherapy can be significantly reduced or even be avoided if the hypoallergenic molecules substantially lack T-cell epitopes.

Truncated Phl p 5 molecules lacking T-cell epitopes are composed of amino acids 93 to 128, 98 to 128, 26 to 53, 26 to 58 or 252 to 283 of Phl p 5 or fragments or sequence variations thereof.

In particular these truncated molecules substantially show no T-cell epitopes and are, nevertheless, able to provoke an appropriate immune response directed against the wild-type allergen.

According to another preferred embodiment of the present invention the hypoallergenic truncated Phl p 5 is composed of amino acids 132 to 162, 217 to 246 or 176 to 212 of Phl p 5 or sequence variations thereof.

These hypoallergenic molecules comprise one or more T-cell epitopes but lack IgE-binding capacity.

Another aspect of the present invention relates to a hypoallergenic molecule derived from Fel d 1 (Genbank Nr. X62477 and X62478) having a C- and/or N-terminal truncation and lacking IgE-binding capacity.

Allergies to animals affect up to 40% of allergic patients. In the domestic environment, allergies to the most popular pets, cats and dogs, are particularly prevalent and connected with perennial symptoms. Animal allergens are present in dander, epithelium, saliva, serum or urine. Exposure to the allergens can occur either by direct skin contact or by inhalation of particles carrying the allergens. The major cat and dog allergens were shown to be present widespread and could even be detected in non-pet owning households and in public places, e.g., schools. This can be attributed to the high and increasing number of households keeping pets in industrialized countries (about 50%) and the high stability of the allergens, which are carried off and distributed.

Fel d 1 was identified as the major cat allergen, which is recognized by more than 90% of cat allergic patients. Fel d 1 represents a 38 kDa acidic glycoprotein of unknown biological function. It consists of two identical non-covalently linked heterodimers, which, again, are composed of two polypeptide chains antiparallely linked by three disulfide bonds. Chain 1 and chain 2 are encoded on different genes, each consisting of 3 exons. Recombinant Fel d 1 (rFel d 1), expressed as a chain 2- to chain 1 fusion protein, has been generated in E. coli. This recombinant Fel d 1 is able to completely mimick the immunological properties of the wild-type allergen.

Peptides derived from the major cat allergen Fel d 1, and lacking IgE-binding capacity are suitable, e.g., for immunotherapy and prophylactic allergy vaccination. These peptides may be comprised in a larger polypeptide or be coupled to a suitable carrier protein such as keyhole limpet hemocyanin (KLH). The Fel d 1-derived synthetic peptides—like the Phl p 5 and allergen-derived peptides disclosed herein—are capable of inducing an IgG response, i.e., the production of so called “blocking antibodies” or “protective antibodies”. These antibodies prevent IgE-binding to the allergen Fel d 1. A significant reduction in allergic symptoms may thus be achieved.

According to a preferred embodiment of the present invention the truncated molecule exhibits reduced T-cell reactivity.

In order to avoid or to significantly reduce late side effects the Fel d 1 derived hypoallergenic molecule exhibits reduced T-cell reactivity as defined in the present invention.

The truncated Fel d 1 is preferably composed of amino acids 1 to 34 or 35 to 70 of chain 1 of Fel d 1, amino acids 1 to 34, 35 to 63 or 64 to 92 of chain 2 of Fel d 1 or sequence variations thereof.

Another aspect of the present invention relates to hypoallergenic molecules being composed of or comprising amino acids 1 to 33, 21 to 51, 42 to 73, 62 to 103 or 98 to 129 of Der p 2, amino acids 1 to 30, 20 to 50, 50 to 80, 90 to 125, 125 to 155 or 165 to 198 of Der p 7, amino acids 1 to 35, 35 to 72, 70 to 100 or 90 to 122 of Der p 21, amino acids 1 to 32, 15 to 48 or 32 to 70 of Clone 30, amino acids 19 to 58, 59 to 95, 91 to 120 or 121 to 157 of Alt a 1, amino acids 31 to 60, 45 to 80, 60 to 96 or 97 to 133 of Par j 2, amino acids 1 to 40, 36 to 66, 63 to 99, 86 to 120 or 107 to 145 of Ole e 1, amino acids 25 to 58, 99 to 133, 154 to 183, 277 to 307, 334 to 363, 373 to 402, 544 to 573, 579 to 608, 58 to 99, 125 to 165, 183 to 224, 224 to 261, 252 to 289, 303 to 340, 416 to 457, 460 to 500 or 501 to 542 of Fel d 2, amino acids 19 to 58, 52 to 91, 82 to 119, 106 to 144 or 139 to 180 of Can f 2, amino acids 19 to 56, 51 to 90, 78 to 118, 106 to 145 or 135-174 of Can f 1, amino acids 27 to 70, 70 to 100 or 92 to 132 of Art v 1, amino acids 31 to 70, 80 to 120, 125 to 155, 160 to 200, 225 to 263, 264 to 300 305 to 350 or 356 to 396 of Amb a 1, amino acids 1 to 34, 35 to 74, 74 to 115, 125 to 165, 174 to 213, 241 to 280, 294 to 333, 361 to 400 or 401 to 438 of Alt a 6, amino acids 1 to 40, 41 to 80, 81 to 120, 121 to 160 of Alt a 2 or fragments or sequence variations thereof.

Another aspect of the present invention relates to a hypoallergenic fusion protein comprising at least two hypoallergenic molecules according to the present invention exhibiting reduced IgE-binding capacity and exhibiting optionally reduced T-cell reactivity.

The hypoallergenic molecules of the present invention which are derived from an allergen and lack IgE-binding capacity may be fused recombinantly or conjugated chemically to each other. As single components (allergen fragments) of the fusion protein/polypeptide also said fusion protein/polypeptide lacks IgE-binding capacity.

The fusion protein according to the present invention may comprise at least two, preferably at least three, more preferably at least four, even more preferably at least five, hypoallergenic molecules according to the present invention. It is, of course, also possible to fuse the hypoallergenic molecules to other peptides, polypeptides and proteins not derived from allergens. These peptides, polypeptides and proteins may when administered to an individual also induce an immunologic reaction or may act as a carrier or exhibit enzymatic activities. The hypoallergenic molecules in the fusion protein according to the present invention may be coupled directly to each other or via a linker which is preferably composed of amino acid residues.

Methods for the production of fusion proteins are well known in the art and can be found in standard molecular biology references such as Sambrook et al. (Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al. (Short Protocols in Molecular Biology, 3rd ed; Wiley and Sons, 1995). In general, a fusion protein is produced by first constructing a fusion gene which is inserted into a suitable expression vector, which is, in turn, used to transfect a suitable hosT-cell. In general, recombinant fusion constructs are produced by a series of restriction enzyme digestions and ligation reactions which result in the desired sequences being incorporated into a plasmid. If suitable restriction sites are not available, synthetic oligonucleotide adapters or linkers can be used as is known by those skilled in the art and described in the references cited above. The polynucleotide sequences encoding allergens and native proteins can be assembled prior to insertion into a suitable vector or the sequence encoding the allergen can be inserted adjacent to a sequence encoding a native sequence already present in a vector. Insertion of the sequence within the vector should be in frame so that the sequence can be transcribed into a protein. It will be apparent to those of ordinary skill in the art that the precise restriction enzymes, linkers and/or adaptors required as well as the precise reaction conditions will vary with the sequences and cloning vectors used. The assembly of DNA constructs, however, is routine in the art and can be readily accomplished by a person skilled in the art.

According to a preferred embodiment of the present invention the molecules are fused to each other in an order differing from the order of the fragments in the wild-type allergen if the at least two molecules are derived from the same allergen.

The fusion protein according to the present invention may comprise at least two hypoallergenic molecules which are derived from the same wild-type allergen. In such a case the single molecules (allergen fragments) are fused to each other in an order differing from the order in the wild-type allergen. Such an approach prevents the re-formation of potential IgE-binding sites/epitopes in the hypoallergenic fusion protein.

Another aspect of the present invention relates to a nucleic acid molecule coding for a hypoallergenic molecule and a fusion protein according to the present invention.

The nucleic acid molecule of the present invention may be employed, e.g., for producing said molecules recombinantly.

Said nucleic acid molecule may—according to another aspect of the present invention—be comprised in a vector.

This vector is preferably an expression vector.

Another aspect of the present invention relates to a vaccine formulation comprising a hypoallergenic molecule, a fusion protein or an antibody according to the present invention.

The formulation further comprises preferably at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.

The use of particular carrier substances such as KLH (keyhole Limpet Hemocyanin) is also among the latest current methods to increase immune responses. The hypoallergenic molecules of the present invention may also be fused or conjugated to viral capsid proteins which may act also as a carrier (see above).

According to a preferred embodiment of the present invention said formulation comprises 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said hypoallergenic molecule or antibody.

The vaccine formulation may be substantially composed as the medicament of the present invention (see above).

Another aspect of the present invention relates to the use of a hypoallergenic molecule, a fusion protein, or an antibody according to the present invention for manufacturing a medicament for the treatment or prevention of an allergy in an individual.

The hypoallergenic molecule, fusion protein and antibody according to the present invention may be used for vaccination of an individual. This vaccination may reduce or prevent the allergic response caused by a wild-type allergen.

According to a preferred embodiment of the present invention said medicament further comprises at least one adjuvant, pharmaceutical acceptable excipient and/or preservative.

The medicament according to the present invention comprises preferably 10 ng to 1 g, preferably 100 ng to 10 mg, especially 0.5 μg to 200 μg of said immunogenic molecule, nucleic acid molecule, vector, host or antibody.

According to another preferred embodiment of the present invention the medicament is administered to an individual in the amount of 0.01 mg/kg body weight to 5 mg/kg body weight, preferably 0.1 mg/kg body weight to 2 mg/kg body weight.

According to a preferred embodiment of the present invention said individual is at risk to get an allergy.

Another aspect of the present invention relates to the use of a hypoallergenic molecule, a fusion protein or an antibody according to the present invention for diagnosing an allergy or monitoring the progress of an allergy therapy in an individual.

The hypoallergenic molecule, fusion protein or antibody according to the present invention may not only be used in medicaments but can also be suitably employed for various diagnostic purposes. For instance, these molecules and fusion proteins may be used for diagnosing an allergy by exposing, e.g., a sample of an individual comprising histamine releasing cells to said polypeptide (see, e.g., Purohit et al., Clin. Exp. Allergy 35 (2005): 186-192). Furthermore, these molecules, fusion proteins and antibodies may be immobilized on a surface in order to form a polypeptide array/chip. Such arrays may be used, e.g., in high throughput screening in order to diagnose an allergy in a number of samples taken from a number of individuals.

The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

FIG. 1A shows a schematic overview of the vector p89VP1.

FIGS. 1B and 1C show the DNA sequence of the multiple cloning site of the pET-17b vector and the 89VP1 encoding gene.

FIG. 1D shows the schematic representation of three possibilities for creating nucleic acid fusions.

FIG. 2 shows a Coomassie blue stained 12% SDS-PAGE gel containing purified 89VP1 his-tagged protein (Lane 1: 5 μg molecular marker; Lane 2-5: 10 μl 89VP1 elution samples).

FIG. 3 shows IgG recognition of 14VP1: Immunoblotting of 14VP1 and controls. Dots are visualized by autoradiography (Lane 1-6: Incubation with 1:500-1:16000 diluted rabbit anti-14VP1 antiserum; Lane 7-12: Incubation with 1:500-1:16000 diluted preimmune serum).

FIG. 4 shows 89VP1-specific IgG1 response in mice. Groups of mice were immunized with different antigens as indicated on top of each box. 89VP1-specific IgG1 titers were measured by ELISA and are expressed as OD values on the y-axis. The results are shown as box plots, where 50% of the values are within the boxes and non-outliers between the bars. The line within the boxes indicates the median values.

FIG. 5 shows Phl p 1-specific IgG1 response in mice. Groups of mice were immunized with different antigens as indicated on top of each box. rPhl p 1-specific IgG1 titers were measured by ELISA and are expressed as optical value (OD 405 nm) on the y-axis. The optical value corresponds to the level of IgG1 antibody in the mouse sera. The results are shown as box plots were 50% of the values are within the boxes and non-outliers between the bars. The line within the boxes indicates the median values.

FIG. 6 shows Timothy grass pollen extract-specific IgG1 response in immunized mice. Groups of mice were immunized with different antigens as indicated on top of each box. Timothy grass pollen extract-specific IgG1 titers were measured by ELISA and are expressed as optical value (OD 405 nm) on the y-axis. The optical value corresponds to the level of IgG1 antibody in the mouse sera. The results are shown as box plots, where 50% of the values are within the boxes and non-outliers between the bars. The line within the boxes indicates the median values.

FIG. 7 shows the mean of % inhibition of patient's IgE-binding to rPhl p 1 by preincubation with antisera against rPhl p 1, r89P5 and KLHP5 of all 19 patients. The % inhibition is shown on the y-axis. The results are shown as bars.

FIG. 8 shows the proliferation of spleen cells of immunized mice. T-cells of immunized mice with different antigens as indicated on top of each box were stimulated with peptide 5, 89VP1(89) and KLH. Medium was used as a reference. At the y-axis the stimulation index is shown. The results are displayed in bars.

FIG. 9 shows IgG1, IgG2, IgG4 and IgA response to 14VP1, 89VP1 and rPhl p 1 detected in human sera by ELISA measurement. 10 patient's sera were tested for four antibodies specific for 89VP1, 14VP1 and rPhl p 1 as indicated on top of each box. Sera taken in autumn and winter are shown on the left hand and right hand of each “bar pair”, respectively. Titers were measured by ELISA and are expressed as optical value (OD 405 nm) on the y-axis. The optical value corresponds to the level of antibody in the human sera. The results are shown as box plots, where 50% of the values are within the boxes and non-outliers between the bars. The line within the boxes indicates the median values.

FIG. 10 shows the detection of anti-89VP1 and anti-rPhl p 1 antibodies in sera of allergic patients. 5 Phl p 1 allergic patient's sera were tested for seven antibodies specific for 89VP1 (left bar of each pair) and rPhl p 1 (right bar of each pair). Titers were measured by ELISA and are expressed as optical value (OD 405 nm) on the y-axis. The optical value corresponds to the level of antibody in the human sera. The results are shown as box plots, where 50% of the values are within the boxes and non-outliers between the bars. The line within the boxes indicates the median values.

FIG. 11 shows anti-14VP1 IgG binding to HRV14 protein extract and purified 14VP1 (Lane 1 and 4: 5 μg Marker; lane 2 and 4: Virus extract; lane 2 and 5: 5 μg 14VP1). Blot A and B was incubated with anti-14VP1 immune serum and preimmune serum, respectively. Bound IgG was detected by Western blotting and visualized by autoradiography.

FIG. 12A shows the neutralization of HRV14 (Lane A (cell control): Cells after preincubation of HRV14 with a dilution of anti-14VP1 immune serum 1:10²-1:10⁸ (row A1-A6); Lane B: Cells after preincubation of HRV14 with a dilution of preimmune serum 1:10²-1:10⁸ (row B1-B6); Lane C: cells after Incubation with HRV14 10 TCD50-10⁶ TCD50 (row C1-C6); D5: cells after incubation with preimmune serum; D6: cells after incubation with immune serum). Cells were plated out in all wells and colored with crystal violet after three days.

FIGS. 12B and 12C show cells stained with crystal violet.

FIG. 13 shows IgG reactivity of anti-peptide antisera with the complete Phl p 5 allergen. IgG reactivities (OD values) to Phl p 5 are shown for 3 serum samples (bleeding 1; preimmune serum; bleedings 2-3: serum samples collected in monthly intervals) obtained from 6 rabbits, each of which was immunized with one of the KLH-conjugated peptides.

FIG. 14 shows allergenic activity of rPhl p 5, and the peptide mix as detected by CD203c expression. Heparinized blood samples from three patients allergic to Phl p 5 were incubated with serial dilutions from 10⁻⁴ to 10 μg/mL of recombinant allergen, an equimolar mix of Phl p 5 derived peptides, anti-IgE or control buffer (co, x-axis). Cells were then stained with CD203c mAb and analyzed for CD203c expression on a FACScan. The stimulation index (SI) is displayed on the y-axis.

FIG. 15 shows identification of Phl p 5-derived peptides which induce low lymphoproliferative responses. PBMCs from timothy pollen allergic patients were stimulated with different concentrations of peptides and, for control purposes, with interleukin-2 (x-axis). Stimulation indices (SI) are indicated on the y-axis.

FIG. 16 shows Fel d 1-derived synthetic peptides which induce Fel d 1 specific IgG immune responses in rabbits. Six rabbits were immunized with the KLH-conjugated Fel d 1-derived synthetic peptides or unconjugated rFel d 1 and 3-4 bleedings were drawn in monthly intervals. IgG reactivities of the preimmune sera and the antisera to ELISA plate-bound rFel d 1 are shown as optical densities (O.D. values, y-axis).

FIG. 17 shows the low allergenic activity of Fel d 1-derived synthetic peptides as determined by CD63 and CD203c expression on basophils of allergic patients. PBMCs from 5 cat allergic patients were incubated with serial dilutions of Fel d 1 (closed boxes) or a mixture of Fel d 1-derived synthetic peptides (open boxes) (x-axis). For patient RR PBMCs were also probed with serial dilutions of Fel d 1-derived synthetic peptides as single components. Induction of expression of surface markers CD203c and CD63 was measured as mean fluorescence intensities, and calculated stimulation indices are shown on the y-axis.

FIG. 18 shows that a treatment with KLH-coupled Bet v 1-derived peptides reduces lymphoproliferative responses to rBet v 1 in sensitized mice. T-cell proliferation was measured in spleen cell cultures after in vitro stimulation with the recombinant birch pollen allergen Bet v 1 (white bars), KLH (black bars), or the peptide mix (grey bars). The bars represent the mean stimulation indices (SI±SD) for the different groups of mice.

FIG. 19 shows the prophylactic vaccination of naive mice with KLH-coupled Bet v 1-derived peptides which reduces lymphoproliferative responses to rBet v 1 after sensitization.

FIG. 20 shows that the prophylactic vaccination of naive mice with KLH-coupled Bet v 1-derived peptides induces a Bet v 1-specific IgG response and primes for the induction of allergen-specific IgG responses by the complete allergen. IgG responses (OD values: y-axis) to Bet v 1 were measured in the four treatment groups at different points of time (x-axis).

FIG. 21 shows a comparison of IgE-reactivity: IgE binding capacity of Phl p 5 derived peptides (1, 2) and variants (1a, 2b) was determined in dot blot assays applying 0.2□g/dot using sera from 7 grass-pollen allergic patients (p1-p7) and the serum from a non-atopic individual (NHS). rPhl p 5 was used as positive control and HSA as negative control. Bound IgE was detected with 125 I-labelled anti-human IgE.

FIG. 22 shows a lymphoproliferative responses of Phl p 5 derived peptides (1, 2) and variants (1a, 2b). PBMCs from grass pollen allergic patients were stimulated with different concentrations of peptides and, for control purposes, with equimolar concentrations of rPhl p 5. Stimulation indices (SI) are indicated on the y-axis.

FIG. 23 shows the cross protection of anti VP1 antibodies.

EXAMPLES Example 1 Construction of Vector p89VP1

Virus stock samples were prepared for RT-PCR by addition of 1 μl of RNase inhibitor (Boehringer GmbH, Germany) to a final concentration of 0.01 U/μl after RNA extraction from cell culture supernatants by QIAamp viral RNA kit (Qiagen, Germany).

Plasmid p89VP1 (FIG. 1A) was constructed by replacing the NdeI/EcoRI fragment of the multiple cloning site of pET-17b with the cDNA sequence encoding for the VP1 protein of human rhinovirus strain 89 (89VP1). The NdeI and EcoRI restriction sites (in pET-17b) were used for insertion of AseI/EcoRI linked 89VP1 with ATG at the 5′ end, six histidin followed by the stop-codon TGA at the 3′ end (FIG. 1B).

The insertion of 89VP1 in pET-17b was confirmed by nucleotide sequencing.

After NdeI/AseI fusion instead of the NdeI site CATAAT was created and could not be cut with any available enzyme. Therefore, the site was mutated to CTTAAG, the restriction site of Afl II. To insert a further allergen fragment, the ACCGTT sequence at the 3′ end was mutated to ACCGGT, the restriction site of AgeI. The amino acid sequences are displayed below the nucleotide sequences of 89VP1. The restriction sites are shown underlined in FIG. 1B.

Said Afl II and AgeI restriction sites were created with the Quick change site mutagenesis Kit (Stratagene).

cDNAs for gene fragments can thus be inserted for easy purification into the remaining restrictions sites either at the 5′ end (using Afl II) or at the 3′ end (using AgeI) of 89VP1 or into both sites as indicated in FIG. 1C. Recombinant allergen fragments will thus be expressed at the N-terminus and/or the C-terminus of the 89VP1.

TABLE I Cloning and mutagenesis primers (5′ to 3′) 89VP1 cloning SEQ ID No. 89VP1 forward CGGAATTCATTAATATGAACCCAGTTGAAAAT- 1 TATATAGATAGTGTATTA 89VP1 reverse CGATTAATTCAGTGGTGGTGGTGGTGGTG- 2 GACGTTTGTAACGGTAA 89VP1 cloning Mutagenesis SEQ ID No. Afl II CTTTAAGAAGGAGATATACTTAAGATGAAC- 3 forward CCAGTTG Afl II CAACTGGGTTCATCTTAAGTATATCTCCTTCT- 4 forward TAAAG AgeI forward CCTGATGTTTTTACCGGTACAAACGTCCACCAC 5 AgeI reverse GTGGTGGACGTTTGTACCGGTAAAAACATCAGG 6

Example 2 Cloning of a Construct Expressing a 89VP1-Allergen Fragment Fusion Protein

The approach described above was exemplified for a C-terminal Phl p 1 allergen fragment, namely peptide 5 (CVRYTTEGGTKTEAEDVIPEGWKADTAYESK; M. Focke et al. FASEB J (2001) 15:2042-4). The peptide 5 DNA sequence was amplified by PCR using the Phl p 1 cDNA (GenBank: X78813) as a template (Primers: Phl p 1 forward 5′-CGCGCTTAAGATGGTCCGCTACACCACCGAGGGC-3′ (SEQ ID No. 7); Phl p 1 reverse 5′-CGCGCTTAAGCTTGGACTCGTAGGCGGTGTCGGC-3′ (SEQ ID No. 8)). The PCR product was inserted into the Afl II restriction site of the p89VP1 vector, and the resulting construct is referred to as vector p89P5 and gene product r89P5.

Example 3 Expression and Purification of 89VP1 Peptide 5-Fusion Protein and 89VP1

In order to achieve expression of 89VP1 or r89P5 (recombinant 89VP1-peptide 5 fusion protein), plasmids were transformed into E. coli BL21 (DE3). Transformed E. coli cells were grown in 250 ml LB medium containing 100 mg/l ampicillin at 37° C. to an optical density (600 nm) of 0.4, and protein expression was induced by addition of isopropyl-beta-D-thiogalactosidase (IPTG) to a final concentration of 1 mM. E. coli cells were harvested after 4 hours by centrifugation at 3500 rpm at 4° C. for 10 min. Purification was performed with the Qiagen protocol using Qiagen Ni-NTA and columns. The cell pellet was resuspended under denaturing conditions in 5 ml 6 M guanidinium hydrochloride for 1 hour. After centrifugation (20 min, 10000×g) the supernatant was incubated with 1 ml Ni-NTA for an additional hour. The suspension was then loaded on a column, washed twice with 5 ml wash buffer (8 M urea pH 6.3) and then eluted with 4 ml elution buffer (8 M urea pH 3.5). Renaturation was achieved after dialysis with decreasing molarity of urea.

Purity and size of the purified protein were analyzed by SDS-PAGE as shown in FIG. 2. Protein bands correlated with the calculated protein size of 33.6 kD. Integrity of the proteins was also confirmed by Western blot analysis, using anti-histidine tag antibody.

Example 4 Detection of 14VP1-Specific Rabbit Antibodies by Immunoblotting

5 μg 14VP1 (VP1 protein of the human rhinovirus strain 14) and controls (Bet v 1, Phl p 5, BSA) were dotted onto nitrocellulose membrane strips. These strips were exposed to a dilution of rabbit anti-14VP1 antiserum (lane 1-6) and preimmune serum (lane 8-12). Bound rabbit IgG antibodies were detected with 125I-labeled donkey anti-rabbit IgG 1:1000 and visualized by autoradiography (FIG. 3).

Dot blot analysis of rabbit anti-14 VP1 serum shows that 14VP1 is strongly immunogenic. IgG antibodies were still detected with a 1:16000 dilution of the antiserum in contrast to the preimmune serum and the Bet v 1, Phl p 5 and BSA controls.

Example 5 89VP1-Specific Antibody Response in Immunized Mice Determined by ELISA

In order to determine the immunogenicity of 89VP1 and its ability to act as a carrier for peptide 5, groups of six week old female balb/c mice (Charles River) were immunized with the following antigens: KLH, KLH maleimide coupled peptide 5 (KLHP5) and KLH EDC coupled peptide 5 (KLHP5edc). The chemical coupling was made with the Imject maleimide activated mcKLH and Imject Immunogen EDC Kit (Pierce). The maleimide group reacts with SH groups and the EDC reacts with carboxyl and amino groups to form stable bonds. Groups of 4 mice were immunized with 89VP1, r89P5 and 89P5edc and 2 mice were immunized with peptide 5 only (5 μg each and mixed 1:2 with aluminium hydroxide). The mice were immunized subcutaneously in approximately three-week intervals and bled from the tail veins. The 89VP1-specific IgG1 antibody level was determined by ELISA.

ELISA plates (Nunc) were coated with 5 μg/ml 89VP1. The mice anti-89VP1, anti-r89P5 and peptide 5 sera were diluted 1:500. Bound IgG1 was detected with rat anti-mouse IgG1 (BD Pharmingen) 1:1000 and then with goat anti rat IgG POX-coupled (Amersham Bioscience) 1:2000. The optical value (OD 405 nm) is displayed on the y-axis and corresponds to the level of IgG1 antibody in the mouse sera (FIG. 4).

KLHP5, KLHP5edc, KLH and peptide 5 were used as controls. IgG1 antibodies were detected with an increasing titer during immunization in mice injected with 89VP1 (89VP1, 89P5edc and r89P5). Rabbits immunized with 89VP1, r89P5 and KLHP5 show the same result.

Example 6 rPhl p 1-Specific Antibody Response in Immunized Mice Determined by ELISA

To evaluate whether immunization with r89P5 will induce IgG antibodies that react with complete Phl p 1, the same method and the same mice sera were used as described in example 5. ELISA plates were coated with 5 μg/ml rPhl p 1 and the IgG1 antibody titer was determined (FIG. 5).

All Phl p 1 derived peptides either coupled to KLH or 89VP1 induced rPhl p 1 specific IgG1 antibodies with increasing responses during immunizations. Rabbits immunized with r89P5 and KLHP5 show the same result.

Example 7 ELISA Detection of Timothy Grass Pollen Extract-Specific IgG1 Antibodies

Immunization of mice and ELISA analysis was performed as described in section 5. Whole timothy grass pollen extract was coated (100 μg/ml) on ELISA plates and the IgG1 antibody titer was determined (FIG. 6).

After three immunizations extract-specific IgG1 antibodies could be detected in mice immunized with peptide 5.

Example 8 Rabbit Anti-r89P5 Antibodies Block Patient's IgE-Binding to rPhl p 1

To determine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to rPhl p 1, ELISA plates were coated with 1 μg/ml rPhl p 1, washed and blocked. The plates were preincubated with 1:100-diluted rabbit anti-peptide (89P5, KLHP5), a rabbit anti rPhl p 1 and, for control purposes, with the corresponding preimmune sera. After washing, plates were incubated with human sera from Phl p 1-allergic patients (1:3 diluted) and bound IgE was detected with mouse anti-human IgE (Pharmingen 1:1000) and then with sheep anti-mouse IgG POX-coupled (Amersham Bioscience) 1:2000. The percentage of inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: 100-OD_(i)/OD_(P)×100.

OD_(i) and OD_(P) represent the extinctions after preincubation with the rabbit immune and preimmune serum, respectively. Table 2 shows the capacity of anti-Phl p 1 peptide antibodies to inhibit the binding of 19 allergic patients' IgE to complete rPhl p 1. FIG. 7 displays the mean inhibition (in %) of all anti-rPhl p 1, anti-r89P5 and anti-KLHP5 immune sera. Anti-peptide sera blocked the IgE-binding much better then rPhl p 1. The ability for inhibition is with 89P5 and KLHP5 almost alike. Table 2 shows the inhibition (in %) of all 19 patients.

TABLE 2 % inhibition of 19 patients' IgE-binding to rPhl p 1 after incubation with rabbit anti-rPhl p 1, r89P5 and anti-KLHP5 antisera % inhibition patient rPhl p 1 r89P5 KLHP5 1 32.343 68.291 68.213 2 29.373 64.915 61.509 3 10.367 59.469 66.270 4 28.087 73.906 71.330 5 13.808 49.358 45.372 6 22.597 66.259 67.331 7 5.375 26.667 18.902 8 22.478 42.612 47.979 9 5.019 39.822 56.837 10 13.756 53.878 63.047 11 26.444 58.430 57.944 12 25.795 67.243 62.458 13 41.330 75.694 79.517 14 35.543 85.714 87.012 15 45.796 84.255 75.185 16 32.641 76.508 77.412 17 26.483 63.171 47.735 18 19.229 85.750 86.642 19 31.142 62.428 71.086

Example 9 T-Cell Proliferation of Mouse Spleen Cells after Antigen Stimulation

Groups of three mice were immunized with KLH, KLHP5 and KLHP5edc. Groups of 4 mice were immunized four times with 89VP1, r89P5 and 89P5edc, and 2 mice were immunized with peptide 5 only (5 μg each). Spleen cells were taken 10 days after the last immunization and single cell cultures were stimulated in triplicates in 96 well plates with peptide 5 (P5), 89VP1, KLH, Con A and a medium as a positive and negative control, respectively. After four days radioactive [³H]thymidine 0.5 μCi was added to each well. Cells were then harvested with a Packard Cell Harvester onto unifilter plates after 15 hours. Cell-associated radioactivity was measured with a beta-counter. Stimulation indices where calculated and are shown at the y-axis. The antigen which was used for stimulation is shown on the x-axis. Each box represents the data of the antigen which was used for immunization of the mice (FIG. 8). All values above two count as positive. The KLH and KLHP5 immunized mice are only positive when stimulated with KLH and the peptide 5 mice are completely negative. The KLHP5edc group is also negative which corresponds to the ELISA results. Cells from r89P5, 89P5edc and 89VP1 immunized mice proliferated only after stimulation with 89VP1. The naive control mouse shows no proliferation in all cases. These results show that T-cell epitopes are provided by the carrier 89VP1 and not by the peptide 5.

Example 10 Detection of 14VP1-, 89VP1- and rPhl p 1-Specific Antibodies in Human Sera Obtained in Autumn and Winter by ELISA

Five human sera of randomly chosen persons were taken in autumn and five in winter. The IgG1, IgG2, IgG4 and IgA antibody level against 14VP1, 89VP1 and rPhl p 1 was determined by ELISA as described in example 5. Human IgG1, IgG2, IgG4 and IgA were detected (BD Pharmingen) 1:1000 with sheep anti mouse IgG POX-coupled (Amersham Bioscience) 1:2000. A high anti-14VP1 and 89VP1 IgG1 titer of sera taken in autumn and winter could be detected (FIG. 9). The anti-rPhl p 1 IgG1 antibody titer was much lower. IgG2, IgG4 and IgA antibodies could be detected in all cases at a very low level. The VP1 proteins of the different HRV strains are closely related and cross-reactivity has been shown in other studies.

Example 11 Anti-89VP1 and Anti-rPhl p 1 Antibodies of Phl p 1 Allergic Patients

Sera of five Phl p 1 allergic patients were taken and an ELISA experiment was performed as described in example 5. ELISA plates were coated with rPhl p 1 and 89VP1 and the specific IgM, IgG1, IgG2, IgG3, IgG4, IgA and IgE antibody titer were determined (FIG. 10). More anti-89VP1 IgG1 antibodies than anti-rPhl p 1 IgG1 antibodies could be detected.

Example 12 Detection of Anti-14VP1 Antibody Binding to the Whole Virus by Western Blot Analysis

The IgG antibody binding of sera of the 14VP1 injected rabbit to the whole virus was confirmed by using the whole HRV14 virus (lane 2 and 5) and 5 μg purified 14VP1 (lane 3 and 6) as control. The virus extract was separated by 12% SDS-Page and blotted onto nitrocellulose membrane. Rabbit anti-14VP1 antiserum (lane 1-3) 1:500 and preimmune serum (lane 4-6) 1:500 were tested for binding to HRV14 and 14VP1. Bound IgG was detected with 125I-labelled donkey anti-rabbit antibody and visualized by autoradiography (FIG. 11).

The binding of 14VP1-antiserum could be detected at the same seize (33.6 kD) as 14VP1.

Example 13 Anti-14VP1 Antibodies Neutralization of Intact Human Rhinovirus 14

The tissue culture dosis 50 (TCD50) of HRV14 was determined. Therefore, a virus dilution from 1:10²-1:10⁸ in MEM-Eagle 1% FCS and 40 mM MgCl₂ was performed and incubated in 24 well plates at 34° C. in a humidified 5% CO₂ atmosphere together with HeLa Ohio cells for three days. A control without the virus was also spread out.

The cytotoxic effect of the virus was visualized with crystal violet after three days and the TCD50 (the dilution where 50% of the cells are dead) was calculated.

Serum dilutions and virus (100TCD50) in row A and B were incubated at 34° C. After 2 hours cells were spread out in all wells. D5 and D6 are serum controls. The experimental schema is shown in FIG. 12A. The neutralization effect of the antibodies was detected after three days with crystal violet (FIG. 12A).

Example 14 Characteristics of Phl p 5-Derived Synthetic Peptides

Peptides were synthesized using Fmoc-strategy with HBTU-activation (0.1 mmol small-scale cycles) on the Applied Biosystems peptide synthesizer Model 433A as described. (Focke et al. Faseb J (2001) 15:2042). After in-depth analysis of the Phl p 5 allergen six Phl p 5-derived peptides ranging from 31 (P1: 3026 Dalton) to 38 (P6: 3853 Dalton) amino acids in length which are rich in solvent-exposed amino acids were prepared (Table 3).

These peptides have isoelectric points between 4.32 and 8.98 and three of them (peptide 3, 4 and 6) may contain human T-cell epitopes.

TABLE 3 Characteristics of non-allergenic Phl p 5-derived synthetic peptides. Position (in relation to the Phl p 5 molecule),  sequence, length, molecular weight (MW), isoelectric point (pI) and presence of T-cell epitopes of the Phl p 5-derived peptides are displayed. The cysteine residue added to facilitate the coupling is marked in bold and underlined. aa aa T-cell position Sequence length MW pI epitope Pept. 1  98-128 C GAASNKAFAEGLSGEP- 32 3026 8.16 - (SEQ ID KGAAESSSKAALTSK No. 9) Pept. 2  26-58 ADLGYGPATPAAPAAGYT- 34 3068 4.37 - (SEQ ID PATPAAPAEAAPAGK C No. 10) Pept. 3 132-162 AYKLAYKTAEGATPEAKY- 32 3482 6.29 + (SEQ ID DAYVATLSEALRI C No. 11) Pept. 4 217-246 C EAAFNDAIDASTG- 31 3236 4.87 + (SEQ ID GAYESYKFIPALEAAVK No. 12) Pept. 5 252-283 TVATA- 33 3501 8.98 - (SEQ ID PEVKYTVFETALKKAITAM- No. 13) SEAQKAAK C Pept. 6 176-212 C AEEVKVIPAGELQVIEK- 38 3853 4.66 + (SEQ ID VDAAFKVAATAANAAPANDK No. 14)

Example 15 Phl p 5-Derived Peptides Lack IgE Reactivity and Allergenic Activity

15.1. Lack of IgE Reactivity

To analyze the IgE reactivity of the six Phl p 5-derived peptides, the isolated as well as KLH-coupled Phl p 5-derived peptides were compared with complete rPhl p 5 regarding IgE-binding capacity by ELISA using sera from 29 grass pollen allergic patients (Table 4).

TABLE 4 Serological characterization of 29 grass pollen allergic patients and a non-allergenic control. Sex, age, total serum IgE levels (kU/L), timothy extract-specific IgE (kUA/L), IgE antibodies specific for rPhl p 5 and the 6 isolated (P1-P6) and KLH-coupled (KLH-P1-KLH-P6) peptides were measured by ELISA and ODs (optical densities) are shown. Dashes indicate the lack of IgE reactivity to the isolated as well as to the KLH-coupled peptides. total IgE IgE timothy rPhl IgE (OD) Patient sex age (kU/L) kUA/L) p 5 P1 P2 P3 P4 P5 P6 1 m 29 140.0 25.90 1.437 — — — — — — 2 m 39 76.2 10.50 0.456 — — — — — — 3 f 29 100.0 33.50 0.699 — — — — — — 4 f 31 261.0 28.10 0.930 — — — — — — 5 m 33 380.0 32.00 0.545 — — — — — — 6 f 31 278.0 37.00 1.720 — — — — — — 7 m 43 128.0 20.70 1.118 — — — — — — 8 f 29 200.0 18.40 0.489 — — — — — — 9 f 34 76.6 18.70 0.571 — — — — — — 10 m 35 144.0 39.30 0.157 — — — — — — 11 f 33 79.2 29.60 0.574 — — — — — — 12 f 30 30.3 10.70 0.350 — — — — — — 13 f 34 106.0 20.80 0.395 — — — — — — 14 f 52 448.0 43.00 1.320 — — — — — — 15 f 25 294.0 95.50 1.638 — — — — — — 16 m 30 471.0 82.60 0.752 — — — — — — 17 m 44 2000.0 100.00 2.500 — — — — — — 18 f 30 168.0 66.60 0.806 — — — — — — 19 m 42 512.0 50.30 1.175 — — — — — — 20 f 28 253.0 54.00 1.954 — — — — — — 21 m 30 315.0 100.00 1.054 — — — — — — 22 f 42 401.0 89.50 2.297 — — — — — — 23 f 28 100.0 82.10 1.802 — — — — — — 24 m 42 52.5 3.52 0.885 — — — — — — 25 m 34 136.0 6.11 2.036 — — — — — — 26 m 30 31.2 9.17 1.909 — — — — — — 27 m 36 24.9 4.34 0.233 — — — — — — 28 f 41 41.5 2.19 0.281 — — — — — — 29 f 51 370.0 90.10 1.296 — — — — — — NHS m 39 0.0 0.00 0.065 — — — — — —

ELISA plates (Nunc Maxisorp, Denmark) were coated with Phl p 5-derived peptides (5 μg/ml) or rPhl p 5 as control (5 μg/ml), washed and blocked. Subsequently, plates were incubated with 1:3 diluted sera from 29 grass pollen allergic patients and from a non-atopic individual overnight at 4° C. Grass pollen allergic patients suffering from allergic rhinoconjunctivitis and/or asthma were selected according to case history indicative for seasonal grass pollinosis and characterized by skin prick testing with timothy pollen extract and serological CAP-FEIA (Pharmacia Diagnostics, Uppsala, Sweden) testing. Total IgE levels in the sera were determined by CAP measurements (Pharmacia). IgE antibodies specific for rPhl p 5 were determined by ELISA. Sera from 29 grass pollen allergic patients and a non-atopic individual were used for IgE competition studies. The grass pollen allergic patients group consisted of 13 males and 16 females with a mean age of 35 years (ranging from 25-51 years) (Table 4).

Bound IgE antibodies were detected with a 1:1000 diluted alkaline-phosphatase-coupled mouse monoclonal anti-human IgE antibody (Pharmingen, CA).

Total IgE levels and grass pollen extract-specific IgE ranged from 24.9-2000 kU/L (mean: 262.7) and 2.2-100 kUA/L (mean: 41.5), respectively. All patients contained rPhl p 5-specific IgE antibodies ranging between 0.157-2.530 OD units (mean: 1.082 OD units), but none of the 29 patients showed IgE reactivity to any of the peptides (P1-P6) or to an equimolar mix of the peptides (OD≦0.08). This result demonstrates that no serum contained detectable IgE antibodies with specificity for any of the six Phl p 5 derived peptides.

15.2. Reduced Allergenic Activity of the Peptides as Detected by CD 203 c Expression on Basophils: Basophil Activation and Flow Cytometry

The upregulation of CD 203 c has been described as a surrogate marker for allergen-induced basophil activation and degranulation (Hauswirth et al., J Allergy Clin Immunol 2002; 110:102). Therefore, the allergenic activity of complete rPhl p 5 allergen and an equimolar mix of peptides by measuring CD 203 c upregulation on basophils of grass pollen allergic patients was compared.

Peripheral blood cells were obtained from 3 allergic donors after informed consent had been given. Heparinized blood aliquots (100 μl) were incubated with serial dilutions of recombinant allergens (10-4 to 10 μg/ml), anti-IgE antibody (1 μg/ml) or control buffer (phosphate-buffered saline=PBS) for 15 minutes at 37° C. After incubation, cells were washed in PBS containing 20 mM EDTA. Cells were then incubated with 10 μl of PE-conjugated CD203c mAb 97A6 for 15 minutes at room temperature (RT). Thereafter, erythrocytes were lysed with 2 mL FACS™ Lysing Solution. Cells were then washed, resuspended in PBS, and analyzed by two-color flow cytometry on a FACScan (Becton Dickinson), using Paint-a-Gate Software. Allergen-induced upregulation of CD203c was calculated from mean fluorescence intensities (MFIs) obtained with stimulated (MFIstim) and unstimulated (MFIcontrol) cells, and expressed as stimulation index (MFIstim: MFIcontrol). An SI of ≧2.0 (≧2-fold upregulation) was considered indicative of a specific response.

As shown in FIG. 14 it was found that complete rPhl p 5 shows a dose-dependent (10-4 to 10 μg/mL) increase in expression of CD203c on peripheral blood basophils in a sensitized individual, whereas an equimolar mix of the peptides shows no effect.

Determination of CD 203c expression on basophils from grass-pollen allergic patients indicates that no allergenic activity can be observed with the Phl p 5 derived peptides.

Example 16 Immunization with Phl p 5 Derived Peptides Induces IgG Antibodies Reactive with rPhl p 5 and Natural Allergens from Different Grass Species

16.1. Recombinant Allergens and Allergen Extracts

Purified recombinant Phl p 5 were expressed in E. coli as described (Vrtala et al. J of Immunol (1993) 151:4773-4781).

Grass pollen from Phleum pratense, Lolium perenne, Poa pratensis, Dactylis glomerata, Secale cereale, Triticum aestivum, Avena sativa, Hordeum vulgare, Anthoxanthum odoratum were obtained from Allergon Pharmacia (Sweden), and an aqueous pollen extract was prepared as described (Vrtala et al., Int Arch Allergy Immunol (1993) 102:160-9.).

16.2. Immunization of Rabbits

HPLC-purified peptides were coupled to KLH (keyhole limpet hemocyanin, MW 4.5×103-1.3×107, Pierce, USA) according to the manufacturers advice and purified using a Conjugation Kit (Sigma, USA).

Rabbits were immunized with each of the KLH conjugated-peptides (200 μg/injection) and, for control purposes, with complete rPhl p 5 using Freunds complete and incomplete adjuvant (Charles River). Serum samples were obtained in four week intervals.

16.3. Reactivity of Rabbit Antibodies with Complete rPhl p 5 and Natural Allergens from Different Grass Species

In order to investigate whether antibodies induced after immunization with KLH-coupled peptides recognize the rPhl p 5, natural Phl p 5 and Phl p 5-related grass pollen allergens from other grass pollen species, ELISA experiments were performed. For ELISA detection, plates (Nunc Maxisorp, Denmark) were coated with pollen allergen extracts (100 μg/ml: Phleum pratense, Lolium perenne, Poa pratensis Dactylis glomerata, Secale cereale, Triticum aestivum, Avena sativa, Hordeum vulgare, Anthoxanthum odoratum) or purified recombinant allergen (5 μg/ml: rPhl p 5). ELISA plates were washed and blocked and subsequently incubated with rabbit anti-peptide antisera and corresponding pre-immunsera diluted 1:2500. Bound rabbit IgG was detected with a HRP-coupled goat anti-rabbit Ig antiserum (Jackson Immunresearch, Pennsylvania). Results represent means of duplicate determination with an error of <5% (FIG. 13, Table 5).

TABLE 5 Crossreactivity of anti-Phl p 5 peptide antisera with rPhl p 5 and natural group 5 allergens from Phleum pratense, Lolium, perenne Poa pratensis, Dactylis glomerata, Secale cereale, Triticum aestivum, Avena sativa, Hordeum vulgare, Anthoxanthum odoratum. IgG reactivities (OD values) of peptide antisera (anti-P1 to anti -P6) to Phl p 5 and pollen extracts from grass pollen are displayed for 6 rabbits which were immunized with KLH-conjugated Phl p 5-derived peptides (P1-P6). Crossreactivity of anti-peptide antisera with rPhl p 5 and grass pollen extracts anti- anti- anti- anti- anti- anti- P1 P2 P3 P4 P5 P6 rPhl p5a 1.115 2.418 1.336 1.600 1.540 2.142 Phleum pratense 0.227 1.155 0.955 0.703 1.138 1.000 Lolium perenne 0.056 1.320 0.834 0.238 0.163 2.500 Poa pratensis 0.070 1.491 1.045 1.880 2.200 2.500 Dactylis glomerata 0.060 0.390 0.728 0.689 0.154 0.657 Secale cereale 0.090 0.292 0.777 0.676 0.162 0.843 Triticum aestivum 0.116 1.076 0.734 0.404 0.570 0.703 Avena sativa 0.150 0.790 1.029 0.551 0.224 1.494 Anthoxanthum 0.114 1.209 1.531 0.827 1.114 1.115 Hordeum vulgare 0.080 1.972 1.150 1.184 0.602 1.513

16.4. Immunization with Phl p 5-Derived Peptides Induces Cross-Reactive IgG Antibodies

Immunization with each of the peptides induced a robust Phl p 5-specific IgG response which became detectable four weeks after the first immunization and increased after the second immunization (FIG. 13). Immunization with peptide 2 induced the highest Phl p 5 specific IgG response followed by peptides 6, 4, 5 and 1 which induced the lowest response. With the exception of anti-peptide 1 antibodies which lacked reactivity with group 5 allergens in Lolium perenne, Poa pratensis, Dactylis glomerata, Secale cereale and Hordeum vulgare, the other peptide antisera cross-reacted with each of the grass pollen extracts tested (Table 5).

Example 17 Immunization with Phl p 5-Derived Peptides Induces IgG Antibodies which Inhibit the Binding of Grass Pollen Allergic Patients IgE to Phl p 5

17.1. Inhibition of Allergic Patients' IgE-Binding to rPhl p 5a by Peptide-Specific IgG

Information regarding the capacity of the peptides to induce blocking antibodies is important since blocking antibodies were shown to play a major role in immunotherapy.

In order to examine the ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE to complete rPhl p 5 ELISA, competition experiments were performed using sera from 29 grass allergic patients.

ELISA plates were coated with rPhl p 5 (1 μg/ml) and preincubated either with a 1:250 dilution of each of the anti-peptide antisera (anti-P1-anti-P6), a mixture of the anti-peptide antisera, an anti-rPhl p 5 antiserum or for control purposes, with the corresponding preimmune sera or a mixture of the preimmune sera. After washing, the plates were incubated with 1:3 diluted sera from 29 grass pollen allergic patients and bound IgE antibodies were detected with the alkaline phosphatase-coupled mouse monoclonal anti-human IgE antibody (Pharmingen). The percentage inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: % inhibition of IgE-binding=100-OD_(I)/OD_(P)×100. OD_(I) and OD_(P) represent the extinctions after preincubation with the rabbit's immune and preimmune serum, respectively. Preincubation of Phl p 5 with peptide-induced rabbit IgG inhibited allergic patients IgE reactivity to a varying degree. The average degree of inhibition of IgE binding ranged from 19.3% for anti-peptide 6 IgG to 28.5% with anti-peptide 1 IgG. Rabbit antibodies raised against complete Phl p 5 induced a mean inhibition of IgE binding of 43.6%.

TABLE 6 Rabbit anti-Phl p 5 peptide antisera inhibit serum IgE-binding of timothy pollen allergic patients to Phl p 5. The percentage inhibition of IgE-binding to Phl p 5 is displayed for each patient after preincubation of Phl p 5 with anti-peptide antisera (anti- P1-anti-P6), with a mix of the six anti-peptide antisera (anti- P1-P6) or with an anti-rPhl p 5 antiserum. The mean percentage inhibition is displayed in the bottom line. % Inhibition of IgE-binding to Phi p 5 with antisera specific for Patient P1 P2 P3 P4 P5 P6 P1-P6 Phl p 5 1 0 5 4 0 0 0 0 nd 2 1 10 4 0 0 0 0 nd 3 28 35 28 39 37 38 46 50 4 33 40 33 42 35 45 54 20 5 0 0 3 8 8 0 9 nd 6 46 34 39 47 47 34 21 56 7 41 48 46 49 50 45 49 60 8 41 8 34 18 39 0 0  8 9 6 0 0 0 0 0 0 nd 10 34 46 30 35 39 42 48 38 11 28 0 32 0 28 9 0 nd 12 33 0 27 4 33 0 0 nd 13 30 31 29 0 29 11 15  0 14 46 14 49 17 47 2 8 15 15 48 44 55 22 46 25 23 72 16 41 0 44 24 41 19 8 28 17 52 71 50 57 49 59 73 82 18 43 17 42 0 32 0 10  0 19 5 17 19 16 7 0 4 nd 20 42 54 43 38 38 41 48 65 21 39 51 46 43 43 43 40 39 22 44 49 44 46 44 40 50 70 23 38 54 40 42 48 40 50 66 24 23 0 15 0 0 0 0 nd 25 0 35 0 8 4 28 43 nd 26 51 26 31 21 24 0 19 nd 27 14 15 3 0 9 11 25 nd 28 9 0 17 0 9 0 0 nd 29 10 44 11 31 21 28 24 73 mean 28.5 25.8 28.2 20.9 27.8 19.3 23.0   43.6 N.d.: not done.

Example 18 Phl p 5-Derived Peptides Induce Low Specific Lymphoproliferative Responses

18.1. Lymphoproliferation Assays

In order to identify peptides with the lowest possible T-cell reactivity to minimize therapy-related side effects, the T-cell reactivity was examined by lymphoproliferation assays. Peripheral blood mononuclear cells (PBMC) were isolated from 2 grass pollen allergic patients by Ficoll (Amersham Pharmacia Biotech, UK) density gradient centrifugation. PBMC (2×10⁵) were cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Denmark) in 200 μl serum-free Ultra Culture medium (BioWhittaker, Rockland, Me.) supplemented with 2 mM L-glutamine (SIGMA, USA), 50 μM beta-mercaptoethanol (SIGMA) and 0.1 mg gentamicin per ml (SIGMA) at 37° C. and 5% CO₂ in a humidified atmosphere. Cells were stimulated with different concentrations of synthetic peptides (1.25, 0.6 and 0.3 μg per well) and, for comparison, with 4 U Interleukin-2 per well (Boehringer Mannheim, Germany) and with medium alone. After 6 d culture 0.5 μCi per well [³H]thymidine (Amersham Pharmacia Biotech) was added and 16 h thereafter incorporated radioactivity was measured by liquid scintillation counting using a microbeta scintillation counter (Wallac ADL, Germany). Mean cpm were calculated from the triplicates, and stimulation indices (SI) were calculated as the quotient of the cpm obtained by antigen or interleukin-2 stimulation and the unstimulated control.

PBMCs from timothy pollen allergic patients were stimulated with different concentrations of synthetic peptides. Stimulation indices with peptides were significantly lower than with IL2. Phl p 5-derived peptides induced low specific lymphoproliferative responses. The lowest response was seen with peptide 5 followed by peptide 4.

Example 19 Characteristics of Fel d 1-Derived Synthetic Peptides

In order to obtain peptides suitable for cat allergy vaccination, five peptides which are 30 to 36 amino acids long and cover the whole molecule were designed according to the known amino acid sequence of Fel d 1.

Peptides were synthesized using a Fmoc (9-fluorenylmethoxy-carbonyl)-strategy with HBTU (2-(1H-benzotriazol-1-yl) 1,1,3,3 tetramethyluronium hexafluorophosphate)-activation (0.1 mmol small-scale cycles) on the Applied Biosystems peptide synthesizer Model 433A (USA). Preloaded PEG-PS (polyethylenglycol polystyrene) resins (0.15-0.2 mmol/g loading) (PerSeptive Biosystems, UK) were used as solid phase to build up the peptides. Chemicals were purchased from Applied Biosystems. Coupling of amino acids was confirmed by conductivity monitoring in a feedback control system. One cystein residue was added to peptides 1, 3, 4, and 5 to facilitate coupling to carriers (Table 7). Peptides were cleaved from the resins with a mixture of 250 μl distilled water, 250 μl triisopropylsilan (Fluka, Switzerland), 9.5 ml TFA for 2 h and precipitated in tert-butylmethylether (Fluka, Switzerland) (Focke 2001). The identity of the peptides was checked by mass-spectrometry and peptides were purified to >90% purity by preparative HPLC (PiChem, Austria).

TABLE 7 Molecular characteristics of Fel d 1-derived synthetic peptides. Position within the native Fel d 1 molecule, amino acid sequence, number of amino acids, calculated molecular weight (MW) and theoretical isoelectric point (pI) of the Fel d 1-derived synthetic peptides are shown. All peptides are soluble in water. Amino acid aa Position sequence length MW pI Pept. 1 chain 1, EICPAVKRDVDLFLTGTP- 35 3911 4.30 SEQ ID aa 1-34 DEYVEQVAQYKALPVVC No. 15 Pept. 2 chain 1, LENARILKNCVDAKMTEEDKEN- 36 4083 4.72 SEQ ID aa 35-70 ALSLLDKIYTSPLC No. 16 Pept. 3 chain 2, VKMAITCPIFYDVFFAVANG- 35 3835 4.56 SEQ ID aa 1-34 NELLLDLSLTKVNAC No. 17 Pept. 4 chain 2, TEPERTAMKKIQDCYVENG- 30 3382 4.93 SEQ ID aa 35-63 LISRVLDGLVC No. 18 Pept. 5 chain 2, CMTTISSSKD- 30 3246 4.78 SEQ ID aa 61-92 CMGEAVQNTVEDLKLNTLGR No. 19

The five Fel d 1-derived synthetic peptides have molecular weights in the range of 3246 to 4083 Dalton and have calculated isoelectric points of from 4.30 to 4.93. All five peptides are watersoluble and Peptides 1, 2 and 3 may contain human T-cell epitopes (Table 7).

TABLE 8 Reduced IgE reactivity of Fel d 1-derived synthetic peptides compared to rFel d 1 cat dander total IgE specific IgE rFel d 1 Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide 5 Patient sex age (kU/l) (kUA/l) (O.D.) (O.D.) (O.D.) (O.D.) (O.D.) (O.D.) 1 f 36 >2000 48.2 2.314 0.112 — — — 0.056 2 m 27 798 62.2 2.255 0.069 0.123 — — 0.140 3 m 33 153 9.48 1.394 — — — — — 4 m 25 122 13.2 1.194 1.998 0.113 0.186 — 0.073 5 f 42 267 42.1 1.793 0.074 — — — 0.677 6 f 35 494 37.0 2.007 — — — — 0.204 7 m 27 129 31.3 2.259 — — — — 0.149 8 m 36 1150 13.5 1.384 — — — — 0.130 9 f 32 580 17.3 0.569 — — — — — 10 f 22 189 4.65 0.553 0.051 — — — 0.057 11 f 53 >2000 >100 2.838 0.504 — — — 0.644 12 f 75 4567 47.3 2.519 — 0.060 — — 0.161 13 m 34 >2000 40.0 1.244 — — — — — 14 m n.d. n.d. 1.99 0.178 — — — — — NHS f 27 <2 <0.35 — — — — — —

Example 20 Fel d 1-Derived Synthetic Peptides have Reduced IgE Reactivity Compared to rFel d 1 and Fel d 1-Derived Synthetic Peptides Lack Allergenic Activity

Serum IgE reactivity to the Fel d 1-derived synthetic peptides in order to identify hypoallergenic peptides suited for vaccination was investigated.

The diagnosis of IgE-mediated cat allergy was based on anamnesis, skin prick testing (Allergopharma, Roinbek, Germany) and measurement of total serum IgE as well as of cat dander-specific serum IgE (CAP-FEIA, Pharmacia Diagnostics, Sweden). Non-allergic persons were included for control purposes.

20.1. IgE-Binding Capacity Measured in ELISA Assays

The IgE-binding capacity of the five Fel d 1-derived synthetic peptides was compared with that of the complete rFel d 1 allergen using sera from 14 cat allergic patients. ELISA plates (Nunc Maxisorb, Denmark) were coated with Fel d 1-derived synthetic peptides or rFel d 1 as control (0.5 μg/well), washed and blocked. Plates were then incubated overnight at 4° C. with 1:5 diluted sera from cat allergic patients and from a non-atopic individual. Bound IgE antibodies were detected with a 1:2500 diluted horse-raddish-peroxidase labeled anti-human IgE antibody (KPL, USA).

Sera from 7 female and 7 male cat allergic patients at the age of 22 to 75 years were subjected to CAP-FEIA determinations. Measured total IgE levels ranged from 122 to >4000 kU/l and cat dander specific IgE levels from 1.99 to >100 kUA/l (Table 7). In ELISA assays the IgE reactivity of all 14 tested sera to the major cat allergen Fel d 1 was confirmed. Results were obtained as optical densities (OD) and ranged from 0.178 to 2.838 OD units. IgE reactivity of the 14 sera to Fel d 1-derived synthetic peptides was measured in the same ELISA assay. It was found that IgE-binding was retained for Peptides 1, 2, 3, and 5. IgE-binding was observed for 6/14 sera to Peptide 1, for 3/14 sera to Peptide 2, for 1/14 sera to Peptide 3 and for 10/14 sera to Peptide 5. Measured OD units were between 0.051 and 1.998 for Peptide 1, between 0.060 and 0.123 for Peptide 2, 0.186 for Peptide 3 and between 0.056 and 0.677 for Peptide 5. In summary, all measured OD units were considerably lower than the respective values measured for the whole Fel d 1 allergen.

This demonstrates that Fel d 1-derived synthetic peptides have a reduced IgE reactivity compared to the whole Fel d 1 allergen. Fel d 1-derived synthetic peptides can therefore be considered hypoallergenic, providing the advantage of reduced IgE-mediated side-effects, when used in SIT.

20.2. Specific Induction of Expression of Surface Markers CD203c and CD63 on Human Basophils (FIG. 17)

Since IgE-binding is a prerequisite but not ample for induction of type 1 allergic reactions that also require cross-link of effector cell bound specific IgE, the actual allergenic activity of Fel d 1-derived synthetic peptides was investigated in basophil activation assays. These assays detect an allergen-specific upregulation of surface markers CD203c and CD63, both recognized as markers for basopil activation (Hauswirth et al. J Allergy Clin Immunol. (2002) 110:102-109).

Heparinized blood samples were taken from 5 cat-allergic patients after informed consent had been given. Blood aliquots (100 μl) were incubated with serial dilutions of rFel d 1, Fel d 1-derived synthetic peptides as single components or as equimolar mixture, anti-IgE antibody or buffer (PBS) for 15 minutes at 37° C. After incubation, cells were washed in PBS containing 20 mM EDTA. Cells were then incubated with 10 μl of PE-conjugated CD203c mAb 97A6 and 20 μl of FITC-conjugated CD63 mAb CLB-gran12 for 15 minutes at room temperature. Thereafter, the samples were subjected to erythrocyte lysis with 2 ml FACS™ Lysing Solution. Cells were then washed, resuspended in PBS, and analyzed by two-color flow cytometry on a FACScan (Becton Dickinson, USA), using Paint-a-Gate Software. Allergen-induced upregulation of CD203c and CD63 was calculated from mean fluorescence intensities (MFIs) obtained with stimulated (MFIstim) and unstimulated (MFIcontrol) cells, and expressed as stimulation index (MFIstim: MFIcontrol). An SI of more than 2.0 (i.e. more than 2-fold upregulation) was considered indicative of a specific (diagnostic) response.

On basophils of all five studied cat-allergic patients (RR, EB, KC, MG and SM) stimulation with rFel d 1 induced an allergen-specific upregulation of surface markers CD203c and CD63. The upregulation of CD203c and CD63 was observed to be dose-dependent for 4/5 patients (RR, KC, MG and SM). For these patients CD203c stimulation indices ranged from 1.1 (SM) to 3.2 (RR) for the lowest tested concentration of 0.001 μg rFel d 1/ml and from 3.6 (KC) to 6.2 (RR) for the highest tested concentration of 10 μg rFel d 1/ml. CD63 stimulation indices determined in the same assay ranged from 1.1 (RR) to 2.0 (MG) for the lowest tested rFel d 1 concentration of 0.001 μg/ml and from 3.9 (RR) to 7.3 (MG) for the highest tested rFel d 1 concentration of 10 μg/ml. For Patient EB 0.001 μg/ml Fel d 1 were already sufficient to induce a high-level upregulation of surface markers CD203c and CD63 preventing an observation of dose-dependency of the surface marker upregulation.

Basophils from all five cat-allergic patients were probed with five increasing concentrations (0.005, 0.05, 0.5, 5 and 50 μg/ml) of an equimolar mix of the five Fel d 1-derived synthetic peptides. Basophils from patient RR were additionally probed with five increasing concentrations of the five single Fel d 1-derived synthetic peptides (0.001, 0.01, 0.1, 1 and 10 μg/ml). Peptides were found to be deficient in upregulating the basophil surface markers CD203c and CD63. Peptides were unable to induce any increased expression of CD203c and CD63 on cells of patient RR, KC and SM. A slight upregulation of CD203c (SI=2.3) and of CD63 (SI=2.5) could be detected for patient MG but only for the highest tested concentration of 50 μg peptide mixture/ml, while the lower concentrations applied had also no stimulating effect. A more pronounced upregulation of CD203c (SI-4.2) and CD63 (SI-4.3) was observed for patient EB but, again, only for the highest tested peptide mixture concentration. In both cases, patient MG and EB, the rate of upregulation after stimulation with peptides was considerably lower than the corresponding values for stimulation with the whole Fel d 1 allergen.

This demonstrates that Fel d 1-derived synthetic peptides provide the advantage of holding a lower allergenic activity than the whole Fel d 1 allergen. This is relevant for a decreased risk of IgE-mediated side-effects when Fel d 1-derived synthetic peptides are used in SIT.

Example 21 Immunization with Fel d 1-Derived Synthetic Peptides Induces IgG Antibodies Reactive with the Whole rFel d 1 Allergen

Fel d 1-derived synthetic peptides were shown to be deficient in IgE-binding. As candidate molecules for vaccination, which aims at the induction of allergen-specific IgG antibodies, peptides must retain specific allergen structures and must still be able to induce an IgG immune response specific for the whole allergen. In order to find out whether Fel d 1-derived synthetic peptides fulfill these requirements, immunization experiments in rabbits were performed.

Rabbits were immunized with uncoupled rFel d 1 and KLH-coupled Fel d 1-derived synthetic peptides. HPLC-purified peptides were coupled to KLH via their cysteine residues, using an Imject Maleimide Activated Immunogen Conjugation Kit (Pierce, USA).

Rabbits (New Zealand White rabbits) were immunized with the immunogens (200 μg/injection) using CFA (first immunization) and IFA (first booster injection after 4 weeks; a second booster injection with incomplete adjuvant was given after 7 weeks) (Charles River Breeding Laboratories, Germany). Rabbits were bled 8 weeks after the first immunization and in four-week intervals thereafter.

The induction of peptide- and rFel d 1-specific antibodies was monitored in ELISA assays. ELISA plates (Nunc Maxisorb, Denmark) were coated with rFel d 1 (0.5 μg/well), washed and blocked. Plate-bound rFel d 1 was then probed in duplicates with 1:1000 diluted rabbit antisera and the corresponding rabbit preimmune sera, and bound IgG was detected with an 1:2000 diluted horseradish-peroxidase labelled goat anti-rabbit antiserum (Jackson ImmunoResearch Inc., USA). Means of duplicates were calculated and showed errors of less than 5%.

Immunization with Fel d 1-derived synthetic peptides induces Fel d 1 reactive IgG antibodies. Eight weeks after the first immunization with each of the five Fel d 1-derived synthetic peptides, IgG antibodies reactive to the whole Fel d 1 allergen could be detected in each of the five rabbit antisera. IgG antibody levels remained at comparable levels in the subsequent bleedings (FIG. 16).

Anti-Peptide 1, anti-Peptide 2, anti-Peptide 4 and anti-Peptide 5 rabbit antisera showed IgG reactivities to Fel d 1 at about the same magnitude than the anti-Fel d 1 rabbit antiserum. Also the anti-Peptide 3 rabbit antiserum showed a distinct but a somewhat lower IgG reactivity to Fel d 1.

This indicates that all 5 Fel d 1-derived synthetic peptides are candidate molecules to induce an Fel d 1 specific IgG antibody response.

Example 22 Fel d 1-Derived Synthetic Peptides Induce Weaker Lymphoproliferative Responses than Fel d 1

Desired candidate molecules for improved SIT do not only offer the advantage of reduced IgE-mediated side effects but also of reduced T-cell mediated side effects. In order to investigate the T-cell activating properties of Fel d 1-derived synthetic peptides, lymphoproliferative assays were performed.

PBMCs were isolated from 7 cat-allergic patients by Ficoll (Amersham Pharmacia Biotech, UK) density gradient centrifugation. PBMC (2×105) were cultured in triplicates in 96-well plates (Nunclone, Nalgene Nunc International, Denmark) in 200 μl serum-free Ultra Culture medium (Cambrex, Belgium) supplemented with 2 mM L-glutamine (Sigma, USA), 50 μM β-mercaptoethanol (Sigma) and 0.1 mg gentamicin per ml (Sigma) at 37° C. using 5% CO₂ in a humidified atmosphere. Cells were stimulated with different concentrations (5, 2.5, 1.25 and 0.6 μg/well) of rFel d 1 and Fe d 1-derived synthetic peptides as single components or as equimolar mixture and, for control purposes, with 4U interleukin-2 or with medium alone. After 6 days of culture, 0.5 μCi per well ³H-thymidine (Amersham Pharmacia Biotech) was added and 16 h thereafter, incorporated radioactivity was measured by liquid scintillation counting using a microbeta scintillation counter (Wallac ADL, Germany), and mean cpm were calculated from the triplicates. The stimulation index (SI) was calculated as the quotient of the cpm obtained by antigen or interleukin-2 stimulation and the unstimulated medium control.

IL-2 stimulated proliferation of PBMC from all 7 tested cat-allergic patients, resulting in stimulation indices of 9.8 for RR, 5.2 for EB, 3.2 for KC, 6.7 for MG, 6.3 for SM, 15.7 for RA and of 13.9 for AR.

Fel d 1-derived synthetic peptides induced lower stimulation indices (Table 9).

TABLE 9 Fel d 1-derived synthetic peptides which on an equimolar basis induce weaker lymphoproliferative responses than Fel d 1 can be identified. PBMCs from 7 cat-allergic patients were stimulated with serial dilutions of rFel d 1 or Fel d 1-derived synthetic peptides as single components. Specific lymphoproliferative responses are shown as stimulation indices. 5 μg/w 2.5 μg/w 1.25 μg/w 0.6 μg/w Patient RR rFel d 1 2.6 1.8 1.5 1.9 Peptide 1 1.9 0.6 1.3 1.5 Peptide 2 2.1 1.3 2.0 1.6 Peptide 3 3.5 2.8 2.0 3.0 Peptide 4 2.5 2.4 1.5 0.8 Peptide 5 1.7 0.9 2.3 0.7 Patient EB rFel d 1 8.2 2.9 1.6 1.5 Peptide 1 1.3 0.9 1.0 1.2 Peptide 2 2.4 1.7 1.8 1.6 Peptide 3 1.1 1.2 1.4 1.7 Peptide 4 3.6 3.6 3.2 2.3 Peptide 5 2.2 2.1 1.4 2.1 Patient KC rFel d 1 0.8 1.2 1.3 5.2 Peptide 1 0.7 1.0 1.1 1.1 Peptide 2 1.2 1.5 1.0 1.1 Peptide 3 0.6 0.5 0.5 0.6 Peptide 4 1.6 1.4 1.3 1.1 Peptide 5 1.3 1.4 0.9 1.4 Patient MG rFel d 1 2.9 2.3 2.3 2.2 Peptide 1 1.8 1.4 1.4 1.1 Peptide 2 1.2 1.3 1.4 0.9 Peptide 3 1.1 0.5 0.6 0.7 Peptide 4 1.1 1.5 1.8 1.0 Peptide 5 1.5 1.2 1.6 0.8 Patient SM rFel d 1 2.3 1.6 1.8 1.1 Peptide 1 1.1 1.0 0.8 1.0 Peptide 2 1.8 1.1 1.3 1.2 Peptide 3 2.6 2.1 2.1 1.5 Peptide 4 1.9 1.6 1.7 1.1 Peptide 5 2.3 1.3 1.4 1.0 Patient RA rFel d 1 3.2 1.2 2.4 1.2 Peptide 1 0.8 0.7 1.3 1.1 Peptide 2 1.2 0.5 1.7 1.6 Peptide 3 2.0 2.3 1.6 0.9 Peptide 4 3.0 1.3 1.1 0.6 Peptide 5 0.4 0.6 0.9 0.9 Patient AR rFel d 1 1.4 0.6 0.9 1.0 Peptide 1 1.0 0.5 1.7 0.7 Peptide 2 0.7 0.6 0.9 0.6 Peptide 3 1.6 1.6 2.1 1.0 Peptide 4 1.0 0.7 0.7 0.6 Peptide 5 0.8 0.5 0.3 0.5

Example 23 IgG Antibodies Induced by Immunization with Fel d 1-Derived Synthetic Peptides Inhibit Binding of Cat-Allergic Patients IgE to the Whole Fel d 1 Allergen

The ability of peptide-induced rabbit Ig to inhibit the binding of allergic patients' IgE antibodies to complete rFel d 1 was examined in ELISA competition assays. ELISA plates (Nunc Maxisorb, Denmark) were coated with rFel d 1 (0.05 μg/well), washed and blocked. Plate-bound rFel d 1 was then preincubated with 1:100 diluted rabbit anti-peptide antisera (single anti-peptide antisera as well as a mixture of anti-peptide antisera were used), rabbit anti-rFel d 1 antiserum, and for control purposes also with the respective rabbit preimmune sera. After the plates had been washed, they were incubated with 1:5 diluted human sera from cat-allergic patients. Bound IgE antibodies were detected with a 1:2500 diluted horse-raddish-peroxidase labeled anti-human IgE antibody (KPL, USA). The percentage inhibition of IgE-binding achieved by preincubation with the anti-peptide antisera was calculated as follows: % inhibition of IgE-binding=100−O.D._(I)/O.D._(P)×100, with O.D._(I) being the measured optical density after preincubation with rabbit immune sera and O.D._(P) with rabbit preimmune sera.

Preincubation of ELISA-plate bound Fel d 1 with the 5 anti-peptide rabbit antisera resulted in inhibition patterns that varied between the 14 different tested sera from cat-allergic patients. Anti-Peptide 1 rabbit antiserum blocked patients' IgE-binding to Fel d 1 for 13/14 tested patients' sera, anti-Peptide 2 rabbit antiserum for 8/14, anti-Peptide 3 rabbit antiserum for 13/14, anti-Peptide 4 rabbit antiserum for 9/14 and anti-Peptide 5 rabbit antiserum for 5/14.

Also the range of inhibition showed variations between the different antisera. Among the single tested anti-peptide rabbit antisera, anti-Peptide 1 rabbit antiserum showed best inhibition rates with inhibitions from 0-55% (average 29%). With anti-Peptide 2 rabbit antiserum inhibition rates of 0-18% (average 5%) could be achieved, with anti-Peptide 3 rabbit antiserum of 0-29% (average 11%), with anti-Peptide 4 rabbit antiserum of 0-24% (average 8%) and with anti-Peptide 5 rabbit antiserum of 0-18% (average 4%).

A mix of all 5 anti-peptide rabbit antisera inhibited patients' IgE-binding to Fel d 1 most efficiently with inhibitions achieved for all patients' sera and inhibition rates of 25-84% (average 59%). These inhibitions were even more pronounced than that achieved by preincubation with the anti-Fel d 3 rabbit antiserum (Table 10).

TABLE 10 Rabbit antisera raised against Fel d 1-derived synthetic peptides inhibit binding of human IgE to Fel d 1. The percentage inhibition of IgE-binding to Fel d 1 achieved by preincubation of Fel d 1 with rabbit antisera are shown for 14 patients and as means. Preincubations were performed with 5 rabbit antisera raised against the 5 Fel d 1-derived synthetic peptides (anti-Peptide 1-5), a mix of the 5 anti-peptide antisera (Mix) and an antiserum raised against Fel d 1 (anti-rFel d 1). anti- anti- anti- anti- anti- Mix of the 5 anti- anti- Patient Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide 5 peptide antisera rFel d 1 1 48 18 29 20 18 78 64 2 24 0 8 0 0 67 43 3 55 11 5 17 8 84 74 4 38 7 11 24 8 66 49 5 10 5 5 12 0 54 48 6 33 0 12 5 0 68 46 7 6 1 10 5 0 58 45 8 44 3 17 10 0 60 53 9 26 17 12 15 16 53 43 10 0 0 10 0 0 31 26 11 38 0 0 0 0 52 56 12 47 0 22 0 7 75 51 13 27 2 8 0 0 56 41 14 16 0 6 5 0 25 25 mean 29 5 11 8 4 59 47

When the anti-Peptide 1 rabbit antiserum was combined with each of the other anti-peptide antisera, the inhibition of allergic patients IgE binding was substantially increased reaching almost values (e.g. anti-peptide 1+4:41%, anti-peptide 1+5:42%) obtained with anti-rFel d 1 antibodies (67%) (Table 11).

TABLE 11 anti-Peptide anti-Peptide anti-Peptide anti-Peptide anti- 1 + anti 1 + anti 1 + anti 1 + anti rFel Patient Peptide 2 Peptide 3 Peptide 4 Peptide 5 d 1 1 61 49 61 63 75 2 24 17 28 28 74 3 60 52 62 57 86 4 43 33 43 40 68 5 17 9 27 30 67 6 37 24 42 46 73 7 26 21 36 34 74 8 51 46 53 55 72 9 40 28 46 43 61 10 16 11 30 34 40 11 45 35 47 45 78 12 52 40 56 59 76 13 29 17 29 32 62 14 7 10 14 16 28 mean 36 28 41 42 67

Example 24 Bet v 1-Derived Peptides Induce Bet v 1-Specific IgG Responses Receiving T-Cell Help from Carrier-Derived T-Cell Epitopes and Reduce Bet v 1-Specific T-Cell Proliferation

Bet v 1-derived, surface-exposed B cell peptides have already been shown to induce a protective Bet v 1-specific IgG response in a mouse model of therapeutic and prophylactic vaccination (Focke M et al. Clin Exp Allergy (2004) 34:1525-1533). In Focke M et al. (2004), 6 Bet v 1-derived peptides were coupled to the carrier molecule KLH before immunization of mice. In the present example it is shown that the Bet v 1-specific IgG response induced with these peptides (Table 1) is driven by the help of T-cell epitopes derived from the carrier but not from the Bet v 1 allergen. It was surprisingly found that none of the Bet v 1-derived peptides having the sequences LFPKVAPQAISSVENIEGNGGPGTIKKISF (SEQ ID No. 20), GPGTIKKISFPEGFPFKYVKDRVDEVDHTN (SEQ ID No. 21) and VDHTNFKYNYSVIEGGPIGDTLEKISNEIK (SEQ ID No. 22) induced relevant T-cell responses, and it could be demonstrated that the majority of T-cell responses was directed against the carrier molecule, KLH (FIGS. 18 and 19). This finding is of great importance for the reduction of side effects during therapeutic vaccination and for reducing the risk of a potential sensitization during prophylactic vaccination. It has been demonstrated in the past that allergen-derived peptides lacking any IgE reactivity but containing allergen-derived T-cell epitopes induced side effects due to T-cell activation. Allergen-derived peptides lacking IgE and T-cell reactivity as exemplified for the Bet v 1 peptides, induce neither IgE nor T-cell mediated side-effects during therapeutic vaccination. When used for prophylactic vaccination, the peptides will induce a Bet v 1-specific protective IgG response without priming of Bet v 1-specific T-cells. This should minimize the risk of pre-priming an allergic immune response through the vaccine which might pave the road for a subsequent allergic sensitization.

In this example the allergen- and carrier-specific T-cell responses in a mouse model of therapeutic and prophylactic allergen vaccination were dissected. Bet v 1-derived peptides 2,3, and 6 (Focke M et al. (2004)) were chosen and tested on whether they contain any of the known Bet v 1-specific T-cell epitopes in BALB/c mice. The mice were immunized as follows (Table 10 shows the sensitization and treatment protocol): Groups of BALB/c mice (n=5) were immunized with 10 μg recombinant Bet v 1 (Biomay, Austria) and/or a mixture of the synthetic Bet v 1-derived peptides 2, 3, and 6 (10 μg of each). Peptides were coupled to KLH as previously described (Focke M et al. (2004)). For immunization, Bet v 1 and the peptide mix were adsorbed to aluminium hydroxide (Alu-Gel-S, Serva, Germany) in a total volume of 150 μl/mouse.

TABLE 12 Sensitization and treatment protocol. Sensitization Therapy groups (n = 5) (rBet v 1) (peptides KLH) no Sensitization/no Therapy (S−/T−) — — Sensitization/no Therapy (S+/T−) day 0, 20, 40 — no Sensitization/Therapy (S−/T+) — day 60, 80, 100 Sensitization/Therapy (S+/T+) day 0, 20, 40 day 60, 80, 100 Prophylaxis Sensitization (peptides KLH) (rBet v 1) no Prophylaxis/Sensitization (P−/S+) — day 60, 80, 100 Prophylaxis/no Sensitization (P+/S−) day 0, 20, 40 — Prophylaxis/Sensitization (P+/S+) day 0, 20, 40 day 60, 80, 100

Allergen-, peptide-, and carrier-specific lymphoproliferation was analyzed in a T-cell proliferation assay. Spleens were removed under sterile conditions and homogenized. After lysis of erythrocytes, cells were washed and resuspended in complete medium (RPMI, 10% fetal calf serum, 0.1 mg/ml gentamycin, 2 mM glutamine). Single cell suspensions were plated into 96-well round-bottom plates at a concentration of 2×10⁵ cells/well and stimulated with concavalin A (0.5 μg/well) as a positive control, rBet v 1 (2 μg/well), KLH (2 μg/well), the peptide mix (0.34 μg of each peptide/well) or the medium alone for 4 days. The cultures were pulsed with 0.5 μCi/well tritiated thymidine for 16 h and harvested. The proliferation responses were measured by scintillation counting. The ratio of the mean proliferation after antigen stimulation and medium control values, i.e. the stimulation index (SI), was calculated.

Interestingly, it could be shown that therapeutic vaccination with Bet v 1-derived peptides could reduce Bet v 1-specific proliferation in Bet v 1 sensitized mice (group S+/T+) compared to the sensitized but untreated group S+/T−. In the sensitized and treated group no peptide-specific proliferation could be measured, but according to the carrier effect, a KLH-specific proliferation was observed. The peptide vaccine alone (group S/T+) induced mainly KLH-specific T-cells, but almost no Bet v 1-specific T-cell response (FIG. 18).

Prophylactic vaccination with the peptides induced almost no Bet v 1-specific proliferation (group P+/S−) compared to the Bet v 1-sensitized group P−/S+ but KLH-specific proliferation. In prophylactically vaccinated and subsequently sensitized mice (group P+/S+) Bet v 1 specific proliferation was remarkably reduced, furthermore, no peptide-specific response could be observed in any mouse group (FIG. 19).

Thus, it could be shown in an allergy mouse model that prophylactic vaccination with carrier-bound allergen-derived B cell peptides did not prime peptide-specific T-cells, almost no allergen-specific but carrier-specific T-cells. Prophylactic vaccination preceding allergic sensitization but also therapeutic vaccination of sensitized mice reduces allergen-specific T-cell proliferation.

Prophylactic treatment with Bet v 1-derived peptides induced Bet v 1-specific IgG responses without help by Bet v 1-specific T-cells. Furthermore, prophylactic treatment increased Bet v 1-specific IgG responses induced by the Bet v 1 allergen already 20 and 40 days after first sensitization (FIG. 20).

These results demonstrate that the peptide vaccine induces a Bet v 1-specific IgG response which can be boosted by allergen exposure.

Example 25 Der p 2-Derived Peptides Showing Reduced IgE Binding Capacity

The IgE binding capacity of Der p 2-derived peptides was determined as described in examples 15.1 and 20.1 employing the peptides according to table 13 and using sera of individuals suffering from house dust mite allergy.

TABLE 13 Der p 2-derived peptides Peptide Position Sequence SEQ ID No. Der p 2 Pep 1  1-33 DQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGK  96 Der p 2 Pep 2 21-51 CHGSEPCIIHRGKPFQLEAVFEANQNSKTAK  97 Der p 2 Pep 3 42-73 EANQNSKTAKIEIKASIEGLEVDVPGIDPNANC  98 Der p 2 Pep 4 62-103 EVDVPGIDPNACHYMKCPLVKGQQYDIKYTWIVP-  99 KIAPKSEN Der p 2 Pep 5 98-129 APKSENVVVTVKVMGDNGVLACAIATHAKIRD 100

The results clearly show that the Der p 2 derived peptides of the present invention exhibit significantly reduced IgE binding capacity.

TABLE 14 Results rDer peptide peptide peptide peptide peptide p 2 1 2 3 4 5 means (n = 50) 1.080 0.010 0.015 0.004 0.031 0.006

Example 26 Variations in the Length of Peptides have No Effect on Peptides' IgE-Binding Capacity, T-Cell Reactivity and Immunogenicity

26.1. Design of Peptides

To study the effect of variation in peptides length on IgE-binding capacity, T-cell reactivity and immunogenicity variants of Phl p 5 derived peptides were designed by increasing the length of peptide 1 (P1) and decreasing the length of peptide 2 (P2) by a few amino-acids (Table 15).

Table 15: Position, sequence, length in number of aminoacids and molecular weight of synthetic Phl p 5 derived peptides (1, 2) and variants thereof (1a, 2b)

TABLE 15 Variants of PHL p 5-derived synthetic peptides Molecular Position Number Weight aa Sequence of aa (MW) Peptide 1 98-128 CGAASNKAFAEGLSGEPKGAAESSSKAALTSK 32 3026 1a 93-128 CFVATFGAASNKAFAEGLSGEPKGAAESSSKAALTSK 37 3592 Peptide 2 26-58 ADLGYGPATPAAPAAGYTPATPAAPAEAAPAGKC 34 3068 2b 26-53 ADLGYGPATPAAPAAGYTPATPAAPAEAC 29 2644

26.2. Lack of IgE-Reactivity

To analyse the IgE-reactivity of Phl p 5 derived peptides 1, 2 and their variants 1a, 2b dot-blot assays were performed applying 0.2 μg peptide/dot and using sera from 7 grass-pollen allergic patients (p1-p7) and the serum from a non-atopic individual (NHS). Bound IgE was detected with 125 I-labelled anti-human IgE (Phadia, Uppsala, Sweden). rPhl p 5 was used as positive control and HSA as negative control. Patients react with rPhl p 5 but not with the peptides and peptide variants (FIG. 21). 26.3. Lymphoproliferative Responses

PBMCs from 2 grass pollen allergic patients were stimulated with different concentrations of Phl p 5 derived peptides 1, 2, their variants 1a, 2b and for control purposes with rPhl p 5. Stimulation indices obtained with the peptides were significantly lower than those obtained with rPhl p 5 (FIG. 22).

26.4. Immunogenicity of Peptide Variants

Rabbits were immunized with KLH-coupled Phl p 5 derived peptides and variants. ELISA experiments were used to measure IgG reactivity of the obtained rabbit antisera to peptides and their variants (Table 16). Immunization with peptides and their variants induced cross-reactive IgG antibodies recognizing the peptide and the corresponding variant.

Table 16: Cross-reactivity of anti-Phl p 5 peptide antisera raised in rabbits by immunization with KLH-conjugated peptides. IgG reactivities of peptide antisera to peptides (1, 2) and variants (1a, 2b) are displayed. No reactivity was observed with preimmune-sera (pre P1, pre P1a, pre P2, pre P2b)

-   -   a. Anti peptide 1 antiserum (anti P1) cross-reacts with peptide         1 variant (1a) and anti peptide 1a antiserum (anti P1a)         cross-reacts with peptide 1.     -   b. Anti peptide 2 antiserum (anti P2) cross-reacts with peptide         2 variant (2b) and anti peptide 2b antiserum (anti P2b)         cross-reacts with peptide 2.

TABLE 16 Cross-reactivity of rabbit-antisera a) pre P1 anti P1 pre P1a anti P1a P1 0.041 0.880 0.052 0.947 P1a 0.038 0.705 0.048 0.859 b) pre P2 anti P2 pre P2b anti P2b P2 0.089 1.168 0.042 1.175 P2b 0.075 0.954 0.053 1.122

26.5. Peptide Induced Rabbit Antisera Inhibit Grass Pollen Allergic Patients' IgE Binding to rPhl p 5

The ability of rabbit anti-peptide 2 and 2b IgGs to inhibit human IgE binding to rPhl p 5 was studied in competition ELISAs. ELISA plate bound rPhl p 5 was preincubated with anti P2, anti P2b and, for control purposes, with anti Phl p 5 antisera. Plates were then exposed to sera from 12 grass pollen allergic patients. The percentage of inhibition of IgE-binding to rPhl p 5 is displayed in Table 17. Anti peptide 2 and anti peptide 2b antisera inhibit patients' IgE binding to rPhl p 5 to the same extent.

Competition ELISAs were also performed with rabbit anti peptide 1 and 1a antisera. In example 17 (Immunization with Phl p 5 derived peptides induces IgG antibodies which inhibit the binding of grass pollen allergic patients IgE to Phl p 5) the anti peptide 1 (P1) antibodies inhibited patients IgE binding to Phl p 5 with a mean inhibition rate of 28.5%. Similar results were obtained with anti peptide 1a antiserum which gave an inhibition rate of 23.7%.

Table 17: Inhibition of patients' IgE binding to rPhl p 5 by anti peptide antisera. Anti peptide 2 and anti peptide 2b antisera inhibit patients' IgE binding to rPhl p 5 to the same extent. ELISA plate bound rPhl p 5 was preincubated with anti P2, anti P2b and, for control purposes, with anti Phl p 5 antisera. Plates were then exposed to sera from 12 grass pollen allergic patients. The percentage of inhibition of IgE-binding to rPhl p 5 is displayed.

TABLE 17 % Inhibition of IgE-binding patient anti P2 anti P2b anti Phl p 5 1 33.38 24.40 84.77 2 52.20 57.40 87.00 3 52.70 54.85 90.81 4 51.44 59.76 78.26 5 43.19 49.15 77.93 6 47.04 52.02 83.68 7 62.67 58.00 76.62 8 52.36 50.27 74.44 9 57.63 50.91 88.13 10 35.10 37.99 75.03 11 44.44 41.24 68.39 12 47.56 45.41 77.34

Example 27 Cross-Protection of Anti-VP1 Antibodies

Human Rhinoviruses Comprise Over Hundred Different Strains. In this neutralization test it is shown that the rhinovirus infection of one strain can also be inhibited by VP1 specific antibodies of another strain. HeLa cells were seeded out in the wells at equal density. 100TCD₅₀ HRV14 was preincubated with dilutions of anti-14VP1- and anti-89VP1 antibodies (undiluted; 1:2-1:32 wells 1-6) and added to the wells in lane A and D, respectively. In lanes B and C 100TCD₅₀ HRV 89 preincubated with dilutions of anti-14VP1- and anti-89VP1 antibodies, respectively, were added to the cells. After 3 days live cells were stained violet. Anti-89VP1 antibodies and anti-14VP1 antibodies block the infection of HRV14 in a comparable manner. The antibodies raised against 14VP1 and 89VP1 also inhibit the infection of HRV89 up to the same concentration. 

1. A hypoallergenic protein consisting of at least one hypoallergenic molecule derived from an allergen, which is fused or conjugated to at least one second non-allergenic protein or fragment thereof. 